Cross-linked peptides containing non-peptide cross-linked structure, method for synthesizing cross-linked peptides, and novel organic compound used in method

ABSTRACT

The purpose of the present invention is to provide a cross-linked peptide containing a novel non-peptide cross-linked structure, and a method for synthesizing the same. A cross-linked peptide having a novel non-peptide cross-linked structure, a useful intermediate for synthesizing the cross-linked peptide, and a method for synthesizing the novel cross-linked peptide and the intermediate are provided. The cross-linked peptide is characterized by having an —NR— bond in the cross-linked structure. By using the method for synthesizing the cross-linked peptide, a cross-link can be freely designed and an change can be freely made to a cross-link.

This application is a national stage application under 35 U.S.C. §371 ofInternational Application No. PCT/JP2012/054895, filed Feb. 28, 2012,entitled “NOVEL CROSS-LINKED PEPTIDES CONTAINING NON-PEPTIDECROSS-LINKED STRUCTURE, METHOD FOR SYNTHESIZING CROSS-LINKED PEPTIDES,AND NOVEL ORGANIC COMPOUND USED IN METHOD,” which claims priority toJapanese Patent Application No. 2011-052107, filed Mar. 9, 2011, thecontents of both of which are hereby incorporated in their entirety byreference.

TECHNICAL FIELD

The present invention relates to a cross-linked peptide containing anovel nonpeptidic cross-linked structure. Also, the present inventionrelates to a method of synthesizing such a cross-linked peptide.Further, the present invention relates to a novel organic compound usedfor synthesis of such a cross-linked peptide.

BACKGROUND ART

Almost all physiological processes are based on molecular recognition ofpeptides or proteins and other biologically active components and thelike. A lot of peptides having important biological functions such ashormones, enzymes, inhibitors, enzyme substrates, neurotransmitters,immunomodulators and the like have been found to date. There areresultantly many studies conducted to develop therapeutic means with apeptide, with understanding physiological effects of active substancescomposed of these peptides.

In development of a peptide as a medicinal product, there are newmethods established for treatments and therapies of diseases correlatedwith peptides, however, in use of a peptide as a medicinal product,problems as described below are generated. That is, a) underphysiological conditions, most peptides are decomposed by specific andnonspecific peptidases, to give low metabolic stability, b) due torelatively large molecular weight thereof, absorption after ingestion ispoor, c) excretion through liver and kidney is fast, and d) since apeptide is structurally flexible and receptors for a peptide can bedistributed widely in an organism, undesired side effects occur innon-targeted tissues and organs.

Except for some examples, relatively small natural peptides (peptidecomposed of 30 to less than 50 amino acids) are present under disorderlyconditions due to a lot of conformations in dynamic equilibrium in adiluted aqueous solution, as a result, the peptides lack in selectivityfor a receptor and become liable to undergo metabolism, thus,determination of a biologically active conformation is difficult. When apeptide itself has a biologically active conformation, namely, whenhaving the same conformation as that under condition linked to areceptor, a reduction in entropy in linking to a receptor is smaller ascompared with a flexible peptide, consequently, an increase in affinityto a receptor is expected. Therefore, there is a need for a biologicallyactive peptide having a uniformly controlled conformation, anddevelopment thereof is important.

There are recently many efforts conducted to develop a peptide mimic ora peptide analog (hereinafter, referred to as “peptide mimic” together)showing a more preferable pharmacological property than that of anatural peptide as the original form thereof. “Peptide mimic” used inthe present specification is a compound which is capable of mimicking(agonistic substance) or blocking (antagonistic substance) at receptorlevel, the biological effect of a peptide, as a ligand of a receptor.For obtaining a peptide mimic as the most possible agonistic substance,factors such as a) metabolic stability, b) excellent bioavailability, c)high receptor affinity and receptor selectivity, d) minimum sideeffects, and the like should be taken into consideration. From thepharmacological and medical standpoint, it is often desirable not onlyto mimic the effect of a peptide at receptor level (agonistic action)but also, if necessary, to block a receptor (antagonistic action). Thesame items as the pharmacological items which should be considered fordesigning a peptide mimic as the above-described agonistic substance canbe applied also to designing of a peptide antagonistic substance.

One example of peptide mimics is development of a peptide having acontrolled conformation. This mimics, as correctly as possible, aconformation linked to a receptor of an endogenic peptide ligand. Whenanalogs of these types are investigated, resistance to a proteaseincreases, and resultantly, metabolic stability rises and selectivityrises, thereby lowering side effects.

Overall control in the conformation of a peptide is possible byrestricting flexibility of a peptide chain by cyclization. Cyclizationof a biologically active peptide not only improves its metabolicstability and selectivity for a receptor but also gives a uniformconformation, thereby enabling analysis of the conformation of apeptide. The cyclization form is the same as that observed in naturalcyclic peptides. Examples thereof include side chain-side chaincyclization, or side chain-end group cyclization. For cyclization, sidechains of amino acids not correlated with receptor recognition can bemutually linked, or can be linked to the peptide main chain. As anotherembodiment, there is head to tail cyclization, and in this case, acompletely cyclic peptide is obtained.

For these cyclization operations, a cross-linking technology isimperative. Typical examples of cyclization include cross-linkages via adisulfide bond (SS bond), an amide bond, a thioether bond and an olefinbond. More specific examples thereof include cyclization by connectingtwo penicillamine residues via a disulfide cross-linkage (Mosberg etal., P.N.A.S. US, 80:5871, 1983), cyclization by forming an amide bondbetween lysine and aspartic acid (Flora et al., Bioorg. Med. Chem. Lett.15 (2005) 1065-1068), a procedure in which an amino acid derivativecontaining a cross-linked portion having a thioether bond introducedpreviously is introduced into a peptide bond and cyclization thereof isperformed in the last condensation reaction (Melin et al., U.S. Pat. No.6,143,722), and cyclization by mutually cross-linking(S)-α-2′-pentenylalanines introduced into the main chain using an olefinmetathesis reaction (Schafmeister et al., J. Am. Chem. Soc., 122,5891-5892, 2000).

A cross-linkage via a disulfide bond, however, will be cleaved by areductase generally present in an organism. Also, a cross-linkage via anamide bond will be cleaved by an enzyme cutting an amide structurepresent in an organism. A thioether bond and an olefin bond needsubstitution of side chains of an amino acid in a peptide elongationprocess, for attaining cyclization thereof.

Also known is a cross-linked structure originating from nitrogenconstituting an amide in the peptide main chain skeleton, as a methodneeding no modification of a side chain of a peptide (Gilon et al.,Biopolymers 31:745, 1991). However, this peptide will be cleaved by anenzyme cutting an amide structure, because of inclusion of an amide bondin this peptide.

Further, known as a cross-linked peptide having a molecular structurecapable of linking to other substituent is a cross-linked peptideutilizing 2,4,6-trichloro[1,3,5]-triazine (Scharn et al., J. Org. Chem.2001, 66, 507-513). In this method, however, the reaction in forming across-linked portion is an aromatic nucleophilic substitution reaction,thereby limiting applicable peptides.

As the analogous peptide, a cross-linked peptide in which a side chainand a carboxy terminus are linked is known (Goodman et al., J. Org.Chem. 2002, 67, 8820-8826). This peptide, however, will be cleaved by anenzyme cutting an amide structure, because of inclusion of an amide bondin this peptide.

A peptide having a controlled conformation is expected to provide a lotof pharmacological use applications. For example, somatostatin is acyclic tetradecapeptide present in both the central nerve system andsurrounding tissues and has been identified as an important inhibitoragainst secretion of a grow hormone from pituitary gland, andadditionally, has functions such as suppression of secretion of glucagonand insulin from spleen, regulation of most gastrointestinal hormones,regulation of release of other neurotransmitters correlated with motoractivity and a recognition process all over the central nerve system,and the like. A cross-linked peptide composed of nine amino acids calleda WP9QY (W9) peptide mimicking the steric structure of a contact sitebetween TNF and a TNF receptor suppresses the inflammation activity ofTNFa, and additionally, is known to suppress bone resorption (Aoki etal., J. Clin. Invest. 2006; 116(6):1525-1534).

There is a study conducted to obtain a peptide mimic having metabolicstability improved by adding to the peptide a structure not present innatural peptides, as the peptide mimic showing a more preferablepharmacological property than that of a natural peptide as the originalform thereof, in addition to a cross-linked peptide having aconformation controlled as described above. For example, resistance toan enzyme is improved by using cross-linkages (a cross-linkage via athioether bond, a cross-linkage via an olefin, and the like) other thanthe above-described natural cross-linking (disulfide cross-linkage).Further, resistance to metabolism in an organism is improved by adding,for example, PEG and the like, to the terminus or the side chain of apeptide.

JP-A No. 2004-59509, compounds described in PCT internationalpublication WO2007/034812, compounds described in PCT internationalpublication WO2007/122847, compounds described in PCT internationalpublication WO2010/104169 and compounds described in PCT internationalpublication WO2010/113939

CITED REFERENCE Patent Documents

-   Patent document 1: U.S. Pat. No. 6,143,722-   Patent document 2: JP Laid-Open Application No. 2004-59509-   Patent document 3: PCT international publication WO2007/034812-   Patent document 4: PCT international publication WO2007/122847-   Patent document 5: PCT international publication WO2010/113939

Non-Patent Documents

-   Non-patent document 1: Mosberg et al., P.N.A.S. US, 80:5871, 1983-   Non-patent document 2: Flora et al., Bioorg. Med. Chem. Lett.    15(2005) 1065-1068-   Non-patent document 3: Schafmeister et al., J. Am. Chem. Soc., 122,    5891-5892, 2000-   Non-patent document 4: Gilon et al., Biopolymers 31:745, 1991-   Non-patent document 5: Scharn et al., J. Org. Chem. 2001, 66,    507-513-   Non-patent document 6: Goodman et al., J. Org. Chem. 2002, 67,    8820-8826-   Non-patent document 7: Aoki et al., J. Clin. Invest. 2006;    116(6):1525-1534

SUMMARY OF THE INVENTION Technical Problem

However, introduction of a functional molecule has often damaged thepharmacological function intrinsically owned by a peptide. There hasbeen desired a cross-linking method capable of producing a cross-linkedpeptide while simply carrying out the elongation reaction of a peptideand forming a cross-linking bond at any site. Further, a cross-linkingmethod has been desired which can optionally perform substitution andother alterations also in the cross-linked structure.

One object of the present invention is to provide a cross-linked peptidecontaining a new cross-linked structure or a peptide mimic having thenew cross-linked structure.

Another object of the present invention is to provide a cross-linkingmethod capable of producing a cross-linked peptide or a peptide mimic,which can perform a peptide elongation reaction in a liquid phase andcan form a cross-linked bond at any site.

Still another object of the present invention is to provide across-linked structure in which substitution and other alterations canbe optionally performed also in the cross-linked structure, and across-linked peptide or a peptide mimic containing such a structure.

Even still another object of the present invention is to provide across-linking method capable of producing a cross-linked peptide or apeptide mimic containing a cross-linked structure in which substitutionand other alterations can be optionally performed also in thecross-linked structure.

The other object of the present invention is to provide a novelcross-linked peptide in which resistance to a peptidase and the like isimproved or a peptide mimic having the novel cross-linked structure.

Solution of Problem

The present inventors have intensively studied to solve theabove-described problems and resultantly succeeded in synthesizing across-linked peptide containing a novel nonpeptidic cross-linkedstructure by using a novel organic compound having a cross-linkedstructure in the molecule, leading to completion of the presentinvention.

That is, the present invention provides novel cross-linked peptides,novel compounds and methods of synthesizing these cross-linked peptides,described below.

The present invention provides:

1. A cross-linked peptide represented by the following chemical formula:

(wherein, X and Y represent each independently an alkylene chain having1 to 12 carbon atoms or an alkylene chain having 1 to 66 carbon atomscontaining at least one —O—, —NH— or —S-bond (optionally substituted bya divalent oxygen atom or sulfur atom), Z represents hydrogen, anoptionally substituted alkyl group having 1 to 30 carbon atoms, anoptionally substituted acyl group having 1 to 36 carbon atoms,polyethylene glycol, a tBoc group, a Fmoc group, a Cbz group or a Nosylgroup, [E] represents a hydrogen atom, an optionally substituted acylgroup having 1 to 6 carbon atoms or a peptide having 1 to 20 residuescomposed of amino acids and/or unnatural amino acids as constituentelements, [G] represents OH, an amino group or a peptide having 1 to 20residues composed of amino acids and/or unnatural amino acids asconstituent elements, [F] represents a peptide having 1 to 20 residuescomposed of amino acids and/or unnatural amino acids as constituentelements (here, the sum of the numbers of amino acids of [E], [F] and[G] is at least 3), and (A) and (B) represent each independently astructure represented by any of the following formula:

(wherein, R₁, R₃ and R₄ represent each independently a hydrogen atom ora methyl group, and R₂ represents a hydrogen atom or a side chain of anamino acid or unnatural amino acid.));

2. The cross-linked peptide according to [1] represented by thefollowing formula:

(wherein, X represents an alkylene chain having 1 to 12 carbon atoms oran alkylene chain having 1 to 66 carbon atoms containing at least one—O— or —S-bond, Y represents an alkylene chain having 1 to 12 carbonatoms or an alkylene chain having 1 to 66 carbon atoms containing atleast one —O—, —NH— or —S-bond (optionally substituted by a divalentoxygen atom or sulfur atom), and Z, [E], [F], [G], R₁, R₂, R₃ and R₄ areas described above.);

3. The cross-linked peptide according to [1] represented by thefollowing formula:

(wherein, X represents an alkylene chain having 1 to 12 carbon atoms oran alkylene chain having 1 to 66 carbon atoms containing at least one—O— or —S-bond, Y represents an alkylene chain having 1 to 12 carbonatoms or an alkylene chain having 1 to 66 carbon atoms containing atleast one —O—, —NH— or —S-bond (optionally substituted by a divalentoxygen atom or sulfur atom), and Z, [E], [F], [G], R₁, R₂, R₃ and R₄ areas described above.);

4. The cross-linked peptide according to [1] represented by thefollowing formula:

(wherein, X represents an alkylene chain having 1 to 12 carbon atoms oran alkylene chain having 1 to 66 carbon atoms containing at least one—O— or —S-bond, and Z, [E], [F], [G], R₁ and R₄ are as described above.Here, Xs, R₁ s or R₄ s in the chemical formula may each be the same ordifferent.);

5. The cross-linked peptide according to [1] represented by thefollowing formula:

(wherein, Y represents an alkylene chain having 1 to 12 carbon atoms oran alkylene chain having 1 to 66 carbon atoms containing at least one—O—, —NH— or —S-bond (optionally substituted by a divalent oxygen atomor sulfur atom), and Z, [E], [F], [G], R₂ and R₃ are as described above.Here, Ys, R₂ s or R₃ s in the chemical formula may each be the same ordifferent.);

6. The cross-linked peptide according to any one of [1] to [5], whereinX is selected from the group consisting of the following chemicalformulae:

(wherein, n represents an integer of 1 to 12, m represents an integer of1 to 24 and 1 represents an integer of 1 to 24.);

7. The cross-linked peptide according to any one of [1] to [6], whereinY is any one selected from the group consisting of the followingcompound Y1, compound Y2, compound Y3, compound Y4 and compound Y5:

(Compound Y1)

(Compound Y2)

(Compound Y3)

(Compound Y4)

(Compound Y5)

(here, [H] is represented by the following formula:

[I] is represented by the following formula:

(here, [J] is represented by the following formula:

[K] is represented by the following formula:

(wherein, o represents an integer of 1 to 12, p represents an integer of1 to 27, q represents an integer of 1 to 24, r represents an integer of1 to 8, s represents an integer of 1 to 16, t represents an integer of 1to 15, u represents an integer of 1 to 11 and W represents O or S.);

8. The cross-linked peptide according to any one of [1] to [5], whereinX is any one selected from the group consisting of the followingchemical formulae:

(wherein, n′ represents an integer of 1 to 7, m′ represents an integerof 1 to 11 and l′ represents an integer of 1 to 12.) and Y is any oneselected from the group consisting of the following chemical formulae:

(wherein, o′ represents an integer of 1 to 8, p′ represents an integerof 1 to 11 and q′ represents an integer of 1 to 12.);

9. The cross-linked peptide according to any one of [1] to [8], whereinZ is an acyl group having 1 to 8 carbon atoms, an unsubstituted orsubstituted alkyl group having 1 to 8 carbon atoms, polyethylene glycolhaving a molecular weight of 100 to 10000 Da represented by—C(═O)—CH₂CH₂(OCH₂CH₂)nOCH₂CH₂OCH₃ or any one selected from the groupconsisting of the following formulae:

(wherein, n represents an integer of 1 to 12, q′ represents an integerof 1 to 12, v represents 1 or 2 and w represents an integer of 1 to 12(here, R₅ is represented by the following formula:

and R₆ is represented by the following formula:

10. The cross-linked peptide according to any one of [1] to [9], whereineach of [E] and [G] represents at least one amino acid and/or unnaturalamino acid and [F] represents at least two amino acids and/or unnaturalamino acids;

11. A compound represented by the following chemical formula:

(wherein, X represents an alkylene chain having 1 to 12 carbon atoms oran alkylene chain having 1 to 66 carbon atoms containing at least one—O— or —S-bond, Y represents an alkylene chain having 1 to 12 carbonatoms or an alkylene chain having 1 to 66 carbon atoms containing atleast one —O—, —NH— or —S-bond (optionally substituted by a divalentoxygen atom or sulfur atom), Z represents hydrogen, an optionallysubstituted alkyl group, having 1 to 30 carbon atoms, an optionallysubstituted acyl group having 1 to 36 carbon atoms, polyethylene glycol,a tBoc group, a Fmoc group, a Cbz group or a Nosyl group, [A], [B], [C]and [D] represent each independently a peptide having 1 to 20 residuescomposed of amino acids and/or unnatural amino acids as constituentelements or a single bond, (a) and (c) represent each independently —NH—or a single bond, (b) and (d) represent each independently —(C═O)— or asingle bond (here, the sum of the numbers of amino acids of [A], [B],[C] and [D] is at least 1, and each of them may have a side chainprotective group.), R₁, R₃ and R₄ represent each independently ahydrogen atom or a methyl group, R₂ represents a hydrogen atom or a sidechain of an amino acid or unnatural amino acid, P₁ and P₃ represent eachindependently an amino protective group or a hydrogen atom, P₂ and P₄represent each independently a —O-ester protective group, a —NH-benzylprotective group, an amino group or a hydroxyl group.);

12. A compound represented by the following chemical formula:

(wherein, X, Z, [A], [B], (a), (b), R₁, R₄, P₁ and P₂ are as describedabove. Here, Xs, [A]s, [B]s, (a)s, (b)s, R₁ s, R₄ s, P₁ s and P₂ s inthe chemical formula may each be the same or different. The sum of thenumbers of amino acids of [A] and [B] is at least 1, and each of themmay have a side chain protective group.);

13. A compound represented by the following chemical formula:

(wherein, Y, Z, [C], [D], (c), (d), R₂, R₃, P₃ and P₄ are as describedabove. Here, Ys, [C]s, [D]s, (c)s, (d)s, R₂ s, R₃ s, P₃ s and P₄ s inthe chemical formula may each be the same or different. The sum of thenumbers of amino acids of [C] and [D] is at least 1, and each of themmay have a side chain protective group.);

14. The compound according to any one of [11] to [13], wherein one ormore terminuses composed of P₁ or P₃ represent a hydrogen atom and oneor more terminuses composed of P₂ or P₄ represent a hydroxyl group;

15. The compound according to any one of [11] to [13], wherein any oneterminus composed of P₂ or P₄ represents a 2,4-alkoxy-substitutedbenzyl;

16. The compound according to [15], wherein the number of carbon atomsof the alkoxy-substituted group is 1 to 60;

17. The compound according to any one of [11] to [16], wherein Xrepresents any one compound selected from the group consisting of thefollowing formulae:

(wherein, n represents an integer of 1 to 12, m represents an integer of1 to 24 and 1 represents an integer of 1 to 24.);

18. The compound according to [17], wherein Y is any one compoundselected from the group consisting of the following compound Y1,compound Y2, compound Y3, compound Y4 and compound Y5:

(Compound Y1)

(Compound Y2)

(Compound Y3)

(Compound Y4)

(Compound Y5)

(wherein, [H] is represented by the following formula:

[I] is represented by the following formula:

(here, [J] is represented by the following formula:

[K] is represented by the following formula:

(wherein, o represents an integer of 1 to 12, p represents an integer of1 to 27, q represents an integer of 1 to 24, r represents an integer of1 to 8, s represents an integer of 1 to 16, t represents an integer of 1to 15, u represents an integer of 1 to 11 and W represents O or S.);

19. The compound according to any one of [11] to [18], wherein each of[A], [B], [C] and [D] represents at least one amino acid and/orunnatural amino acid;

20. A method of synthesizing a cross-linked peptide represented by thefollowing formula:

(wherein, X and Y represent each independently an alkylene chain having1 to 12 carbon atoms or an alkylene chain having 1 to 66 carbon atomscontaining at least one —O—, —NH— or —S-bond (optionally substituted bya divalent oxygen atom or sulfur atom), Z represents hydrogen, anoptionally substituted alkyl group having 1 to 30 carbon atoms, anoptionally substituted acyl group having 1 to 36 carbon atoms,polyethylene glycol, a tBoc group, a Fmoc group, a Cbz group or a Nosylgroup, [E] represents hydrogen, an acetyl group or a peptide having 1 to20 residues composed of amino acids and/or unnatural amino acids asconstituent elements, [G] represents OH, an amino group or a peptidehaving 1 to 20 residues composed of amino acids and/or unnatural aminoacids as constituent elements, [F] represents a peptide having 1 to 20residues composed of amino acids and/or unnatural amino acids asconstituent elements (here, the sum of the numbers of amino acids of[E], [F] and [G] is at least 3), R₁, R₃ and R₄ represent eachindependently a hydrogen atom or a methyl group, and R₂ represents ahydrogen atom or a side chain of an amino acid or unnatural aminoacid.), comprising the following steps (a) to (g):

(a) a step of preparing a first component (A), comprising the followingsteps,

-   -   (a-1) a step of, if necessary, condensing a carboxyl protective        group with amino acids or a peptide constituting a partial        peptide sequence of the cross-linked peptide, and further, if        necessary, elongating the condensed group,    -   (a-2) a step of reacting the N terminus side of the peptide or        amino acid synthesized or the carboxyl protective group with an        amino acid derivative containing in the side chain a linker        forming part of a cross-linkage of the cross-linked peptide, to        synthesize the peptide having the linker in the side chain or        the amino acid derivative having the carboxyl group protected,    -   (a-3) a step of, if necessary, further performing a peptide        elongation reaction to elongate the peptide, and    -   (a-4) a step of, if necessary (that is, when the linker end is        not reactive), converting the functional group of the linker        into a form with which the linker can be subjected to the        subsequent cross-linkage forming reaction,

(b) a step of preparing a second component (B), comprising the followingsteps,

-   -   (b-1) a step of condensing a carboxyl protective group with        amino acids or a peptide constituting a partial peptide sequence        of the cross-linked peptide, and if necessary, elongating the        condensed group,    -   (b-2) a step of reacting the N terminus of the peptide or the        amino acid derivative synthesized with a compound containing a        linker forming part of a cross-linkage of the cross-linked        peptide, to synthesize a peptide or an amino acid derivative        having a secondary amine at the N terminus containing the        linker,    -   (b-3) a step of, if necessary, further performing a peptide        elongation reaction to elongate the peptide, and    -   (b-4) a step of, if necessary (that is, when the linker end is        not reactive), converting the functional group of the linker        into a form with which the linker can be subjected to the        subsequent cross-linkage forming reaction,

(c) a step of linking (cross-linking) the first component (A) and thesecond component (B) by the Mitsunobu reaction, a reductive aminationreaction or the Aza-Wittig reaction and the subsequent reductionreaction, to prepare an intermediate having a structure in which the twocomponents are linked via a secondary amine or a tertiary amine,

(d) a step of, if necessary, deprotecting a protective group at the Nterminus of the first component (A) or the second component (B) and/or aprotective group at the C terminus of the first component (A) or thesecond component (B),

(e) a step of condensing the peptide N or C terminus of one componentwith the peptide C or N terminus of another component to form a peptidebond (peptide chain),

(f) a step of, if necessary, post-processing the cross-linked peptide byany method known to those skilled in the art, and

(g) a step of, if necessary, deprotecting the protective group;

21. A method of synthesizing the following cross-linked peptide:

(X, Y, Z, [E] [F], [G], R₁, R₂, R₃ and R₄ are as described above.)comprising the following steps (a) to (g):

(a) a step of preparing a first component (A1) comprising the followingsteps,

-   -   (a-1) a step of, if necessary, condensing a carboxyl protective        group with amino acids or a peptide constituting a partial        peptide sequence of the cross-linked peptide, and further, if        necessary, elongating the condensed group,    -   (a-2) a step of reacting the N terminus side of the peptide or        amino acid synthesized or the carboxyl protective group with an        amino acid derivative containing in the side chain a linker        forming part of a cross-linkage of the cross-linked peptide, to        synthesize the peptide having the linker in the side chain or        the amino acid derivative having the carboxyl group protected,    -   (a-3) a step of, if necessary, further performing a peptide        elongation reaction to elongate the peptide, and    -   (a-4) a step of, if necessary (that is, when the linker end is        not reactive), converting the functional group of the linker        into a form with which the linker can be subjected to the        subsequent cross-linkage forming reaction,

(b) a step of preparing a second component (A2), comprising the samesteps as the above-described steps (a-1) to (a-4),

(c) a step of linking (cross-linking) the first component (A1) and thesecond component (A2) by the Mitsunobu reaction, a reductive aminationreaction or the Aza-Wittig reaction and the subsequent reductionreaction, to prepare an intermediate having a structure in which thefirst component (A1) and the second component (A1) are linked via asecondary amine or a tertiary amine,

(d) a step of, if necessary, deprotecting a protective group at the Nterminus of the first component (A1) or the second component (A2) and/ora protective group at the C terminus of the first component (A1) or thesecond component (A2),

(e) a step of condensing the peptide N or C terminus of the firstcomponent (A1) with the peptide C or N terminus of the second component(A2) to form a peptide bond (peptide chain),

(f) a step of, if necessary, post-processing the cross-linked peptide byany method known to those skilled in the art, and

(g) a step of, if necessary, deprotecting the protective group;

22. A method of synthesizing the following cross-linked peptide:

(X, Y, Z, [E], [F], [G], R₁, R₂, R₃ and R₄ are as described above.),comprising the following steps (a) to (g):

(a) a step of preparing a first component (B1), comprising the followingsteps,

-   -   (a-1) a step of condensing a carboxyl protective group with        amino acids or a peptide constituting a partial peptide sequence        of the cross-linked peptide, and if necessary, elongating the        condensed group,    -   (a-2) a step of reacting the N terminus of the peptide or amino        acid synthesized with a compound containing a linker forming        part of a cross-linkage of the cross-linked peptide, to        synthesize a peptide or an amino acid derivative having a        secondary amine at the N terminus containing the linker,    -   (a-3) a step of, if necessary, further performing a peptide        elongation reaction to elongate the peptide, and    -   (a-4) a step of, if necessary (that is, when the linker end is        not reactive), converting the functional group of the linker        into a form with which the linker can be subjected to the        subsequent cross-linkage forming reaction,

(b) a step of preparing a second component (B2), comprising the samesteps as the above-described steps (a-1) to (a-4),

(c) a step of linking (cross-linking) the first component (B1) and thesecond component (B2) by the Mitsunobu reaction, a reductive aminationreaction or the Aza-Wittig reaction and the subsequent reductionreaction, to prepare an intermediate having a structure in which thefirst component (B1) and the second component (B2) are linked via asecondary amine or a tertiary amine,

(d) a step of, if necessary, deprotecting a protective group at the Nterminus of the first component (B1) or the second component (B2) and/ora protective group at the C terminus of the first component (B1) or thesecond component (B2),

(e) a step of condensing the peptide N or C terminus of the firstcomponent (B1) with the peptide C or N terminus of the second component(B2) to form a peptide bond (peptide chain),

(f) a step of, if necessary, post-processing the cross-linked peptide byany method known to those skilled in the art, and

(g) a step of, if necessary, deprotecting the protective group;

23. A method of synthesizing the cross-linked peptide according to anyone of [20] to [22], wherein the step (c) is carried out under conditionin which at least one of the first component and the second component islinked to the carboxyl protective group as a peptide supporting body,wherein the peptide supporting body is an alkoxy-substituted benzylselected from the group consisting of a 2,4-substituted benzyl alcohol,a 3,5-substituted benzyl alcohol, a 3,4,5-substituted benzyl alcohol anda 2,4,5-substituted benzyl alcohol;

24. The method of synthesizing the cross-linked peptide according to[23], wherein the number of carbon atoms of the alkoxy substituent ofthe 2,4-substituted benzyl alcohol used as the peptide supporting bodyis 1 to 60;

25. A cross-linked peptide represented by the following chemicalformula:

(wherein, Z₁ and Z₃ represent each independently an unsubstituted orsubstituted acyl group having 1 to 36 carbon atoms, an unsubstituted orsubstituted alkyl group having 1 to 30 carbon atoms or a polyethyleneglycol having a molecular weight of 100 to 20000 Da represented by—C(═O)—CH₂CH₂(OCH₂CH₂)_(n)OCH₂CH₂OCH₃, and Z₂ represents a hydroxylgroup, an amino group, an unsubstituted or substituted monoalkylaminogroup having 1 to 30 carbon atoms or a polyethylene glycol having amolecular weight of 100 to 20000 Da represented by —NH—CH₂CH₂(OCH₂CH₂)_(n)OCH₂CH₂OCH₃.); and

26. A cross-linked peptide represented by the following formula:

(wherein, Ac represents an acetyl group, and the polyethylene glycol hasa number-average molecular weight of 500 to 2000 Da).

Advantageous Effects of Invention

In one object of the present invention, amino acids at any positions ina peptide chain can be cross-linked via a linker to synthesize across-linked peptide having a cross-linkage at any position.

In another object of the present invention, a novel cross-linked peptidecan be provided in which the cross-linked portion of the cross-linkedpeptide has a new structure represented by —X—NZ—Y— (here, X, Y and Zare as defined above).

In still another object of the present invention, a peptide mimic can beprovided, and the peptide mimic provided by the present invention canmanifest different biological characteristics from those of peptideshaving a natural cross-linked structure, for example, resistance to apeptidase, and the like.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 shows a schematic view of a synthesis example of a component usedin an intermediate of the present invention.

FIG. 2 shows a schematic view of a synthesis example of a W9cross-linked peptide mimic as a cross-linked peptide of the presentinvention from an intermediate of the present invention.

FIG. 3 shows a schematic view of a synthesis example of another W9cross-linked peptide mimic (Bdev-14) of the present invention.

FIG. 4 shows a partial schematic view of a synthesis route of asynthesis example of still other W9 cross-linked peptide mimics(Bdev-19, -20) of the present invention.

FIG. 5 shows a schematic view of a synthesis example of still other W9cross-linked peptide mimics (Bdev-19, -20) of the present invention.

FIG. 6 shows a schematic view of a synthesis example of still another W9cross-linked peptide mimic (Bdev-21) of the present invention.

FIG. 7 shows a schematic view of a synthesis example of still another W9cross-linked peptide mimic (Bdev-25) of the present invention.

FIG. 8 shows a schematic view of a synthesis example of still other W9cross-linked peptide mimics (Bdev-27 to 30) of the present invention.

FIG. 9 shows a schematic view of a synthesis example of still anothercross-linked peptide mimic (Bdev-31) of the present invention.

FIG. 10 shows a partial schematic view of a synthesis route of asynthesis example of still another cross-linked peptide mimic (Bdev-32)of the present invention.

FIG. 11 shows a partial schematic view of a synthesis route of asynthesis example of still another cross-linked peptide mimic (Bdev-32)of the present invention.

FIG. 12 shows a partial schematic view of a synthesis route of asynthesis example of still another cross-linked peptide mimic (Bdev-32)of the present invention.

FIG. 13 shows a partial schematic view of a synthesis route of asynthesis example of still another cross-linked peptide mimic (Bdev-32)of the present invention.

FIG. 14 shows a partial schematic view of a synthesis route of asynthesis example of still another cross-linked peptide mimic (Bdev-32)of the present invention.

FIG. 15 shows a partial schematic view of a synthesis route of asynthesis example of still another cross-linked peptide mimic (Bdev-32)of the present invention.

FIG. 16 shows a partial schematic view of a synthesis route of asynthesis example of still another cross-linked peptide mimic (Bdev-32)of the present invention.

FIG. 17 shows a partial schematic view of a synthesis route of asynthesis example of still another cross-linked peptide mimic (Bdev-33)of the present invention.

FIG. 18 shows a partial schematic view of a synthesis route of asynthesis example of still another cross-linked peptide mimic (Bdev-33)of the present invention.

FIG. 19 shows a partial schematic view of a synthesis route of asynthesis example of still another cross-linked peptide mimic (Bdev-33)of the present invention.

FIG. 20 shows a partial schematic view of a synthesis route of asynthesis example of still another cross-linked peptide mimic (Bdev-33)of the present invention.

FIG. 21 shows a synthesis example of a W9 peptide mimic having athioether cross-linkage as a reference example.

FIG. 22 shows a synthesis example of a W9 peptide mimic having an olefincross-linkage as a reference example.

DESCRIPTION OF EMBODIMENTS

In the present specification, “cross-linked peptide” means a peptidechain in which side chains of amino acids in the main chain or terminusgroups of the peptide chain constitute a circular form, andcircularization is caused by side chain-side chain cyclization, sidechain-terminus group cyclization or terminus group-terminus groupcyclization, and as the cyclization, side chains and/or terminus groupsmay be directly linked or may be linked via a cross-linked structurehaving any length between them, and the cross-linked peptide of thepresent invention is characterized by having a —NR— bond in thecross-linked structure. R will be illustrated in detail in the presentspecification, together with other features of the present invention.

In the present specification, “amino acid” is used in a meaningincluding natural L-configured amino acids, and examples thereof includeglycine, alanine, leucine, proline, phenylalanine, tyrosine, methionine,serine, threonine, cystine, cysteine, aspartic acid, glutamic acid,asparagine, glutamine, lysine, arginine, hydroxylysine, histidine,tryptophan, valine, β-alanine and the like, and α-amino acids in themare L-configured. In the present specification, “unnatural amino acid”includes, but not limited to, D-configured bodies and racemic bodies ofthe above-described natural amino acids, L-configured bodies,D-configured bodies and racemic bodies of hydroxyproline, norleucine,ornithine, naphthylalanine, nitrophenylalanine, chlorophenylalanine,fluorophenylalanine, thienylalanine, furylalanine, cyclohexylalanine,homoarginine, homoserine, 3-amino-2-benzylpropionic acid, N-Me typeamino acid and the like, and derivatives thereof. A lot of unnaturalamino acids or amino acid derivatives are well known in the art, andincluded in “unnatural amino acid” referred to in the presentspecification.

In the present specification, when referred to “the number of aminoacids”, it means the number of amino acids of amino acids and/orunnatural amino acids, and in a peptide containing both amino acids andunnatural amino acids, it means the total number thereof.

1. Cross-Linked Peptide of the Present Invention

The cross-linked peptide (or peptide mimic) having a nonpeptidiccross-linked structure of the present invention has a structuredescribed below.

More specific embodiments of the cross-linked peptide (or peptide mimic)having a nonpeptidic cross-linked structure of the present invention hasthe following structures.

The definitions of marks in the above-described chemical formulae (P),(P-1), (P-2), (P-3) and (P-4) are as described below.

X represents an alkylene chain having 1 to 12, preferably 1 to 8, morepreferably 1 to 4 carbon atoms or preferably a methylene chain thereof,or an alkylene chain having 1 to 66, preferably 1 to 30, more preferably1 to 16 carbon atoms containing at least one —O— or —S-bond orpreferably a methylene chain thereof. More preferably, X is selectedfrom the following formulae.

(wherein, n represents an integer of 1 to 12, preferably an integer of 1to 7, further preferably an integer of 1 to 4. m represents an integerof 1 to 24, preferably an integer of 1 to 11, further preferably aninteger of 1 to 7. l represents an integer of 1 to 24, preferably aninteger of 1 to 12, further preferably an integer of 1 to 8). X is, morepreferably, selected from the above-described chemical formulae (X-1),(X-2), (X-3) and (X-5), and most preferably, X is (X-1).

Y represents an alkylene chain having 1 to 12, preferably having 1 to 8,more preferably having 1 to 4 carbon atoms or preferably a methylenechain thereof, or an alkylene chain having 1 to 66, preferably having 1to 30, more preferably having 1 to 16 carbon atoms containing at leastone —O—, —NH— or —S-bond (optionally substituted by a divalent oxygenatom or sulfur atom) or preferably a methylene chain thereof. Morepreferably, Y is selected from the following formulae. (Compound Y1)Hereinafter, these are called Y1-1 and Y1-2 in descending order.

(Compound Y2) Hereinafter, these are called Y2-1 to Y2-4 in descendingorder.

(Compound Y3) Hereinafter, these are called Y3-1 to Y3-9 in descendingorder.

(Compound Y4) Hereinafter, these are called Y4-1 to Y4-5 in descendingorder.

or(Compound Y5)

(wherein, [H] is represented by the following formula:

[I] is represented by the following formula:

(here, [J] is represented by the following formula:

[K] is represented by the following formula:

(wherein, o represents an integer of 1 to 12, preferably an integer of 1to 8, more preferably an integer of 1 to 6. p represents an integer of 1to 27, preferably an integer of 1 to 11, preferably an integer of 1 to7. q represents an integer of 1 to 24, preferably an integer of 1 to 12,more preferably an integer of 1 to 8. r represents an integer of 1 to 8,preferably an integer of 1 to 4. s represents an integer of 1 to 16,preferably an integer of 1 to 11, more preferably an integer of 1 to 6.t represents an integer of 1 to 15, preferably an integer of 1 to 10,more preferably an integer of 1 to 4. u represents an integer of 1 to11, preferably an integer of 1 to 7, more preferably an integer of 1 to3. W represents O or S).

More preferably, Y is selected from (Y1-1), (Y1-2), (Y2-1), (Y2-2),(Y2-3), (Y4-1), (Y4-2) and (Y4-4), most preferably, Y is selected from(Y1-1) and (Y1-2).

Z represents hydrogen, an optionally substituted alkyl group having 1 to30 carbon atoms, an optionally substituted acyl group having 1 to 36carbon atoms, polyethylene glycol, a tBoc group, a Fmoc group, a Cbzgroup or a Nosyl group.

Here, the substituent is not particularly restricted providing that iscan be linked by an organic reaction, and can be optionally selecteddepending on the object of the cross-linked peptide to be synthesized.Examples thereof include, but not limited to, a carboxyl group, analdehyde group, an amino group, a thiol group, a maleimide group, aN-hydroxysuccinimide ester group, a pentafluorophenyl ester group, anisocyanate group, a thioisocyanate group, an acyl group, an alkyl group,an alkynyl group, an alkenyl group or an alkoxy group, or polyethyleneglycol and the like.

Preferably, Z is an optionally substituted acyl group having 1 to 36carbon atoms (for example, an acetyl group), an optionally substitutedalkyl group having 1 to 30 carbon atoms, a polyethylene glycol having amolecular weight of 100 to 20000 Da, a tBoc group, a Fmoc group, a Cbzgroup or a Nosyl group, or any one selected from the group consisting ofgroups represented by the following formula.

(wherein, n represents an integer of 1 to 12, preferably of 1 to 8, morepreferably of 1 to 6, q′ represents an integer of 1 to 12, preferably of1 to 8, more preferably of 1 to 4, v represents 1 or 2, w represents aninteger of 1 to 12, preferably of 1 to 8, more preferably of 1 to 4(here, R₅ is represented by the following formula:

R₆ is represented by the following formula:

Further preferably, Z is hydrogen, an optionally substituted acyl grouphaving 1 to 16 carbon atoms, a polyethylene glycol having a molecularweight of 1000 to 20000 Da, a tBoc group, a Fmoc group, a Cbz group or aNosyl group.

In the cross-linked peptide of the present invention, it is possible tointroduce a functional group or a substance capable of contributing tothe stability and physiological activity of a peptide and the like intoZ, thereby improving the physiological activity of a peptide andallowing a peptide to have a new function, and such a peptide is alsoincluded in the scope of the present invention.

The above-described (A) and (B) represent each independently anystructure represented by the following formula:

The above-described R₁, R₃ and R₄ represent each independently ahydrogen atom or a methyl group, and R₂ represents a hydrogen atom, or aside chain of an amino acid or unnatural amino acid. When R₂ is a sidechain of an amino acid or unnatural amino acid, its kind is notparticularly restricted, and in the case of, for example, serin, R₂ isCH₂OH.

The above-described [E] represents a peptide composed of any amino acidsand/or unnatural amino acids, or a hydrogen atom or an optionallysubstituted acyl group having 1 to 6 carbon atoms, preferably representsat least one amino acid and/or unnatural amino acid, or a hydrogen atomor an acetyl group. The above-described [G] represents a peptidecomposed of any amino acids and/or unnatural amino acids, or a hydroxylgroup or an amino group. The length of a peptide can be arbitrarilyselected in the present invention, and for the purpose of synthesis of apeptide mimic of a peptide having physiological activity and from thestandpoint of a peptide synthesis technology, the number of amino acidsand/or unnatural amino acids of [E] or [G] is 1 to 20, preferably 1 to10. However, the number of amino acids is not limited to this.

Further various modification groups can be attached to the terminus of[E] and/or [G], and also these embodiments are included in the scope ofthe cross-linked peptide of the present invention. Such a cross-linkedpeptide can be obtained by attaching a modification group to theterminus of [E] and/or [G] using a known method after synthesis of theabove-described cross-linked peptide, or by using an amino acidconstituting the terminus of [E] and/or [G] carrying a modificationgroup in a synthesis process of a cross-linked peptide of the presentinvention.

In contrast, the above-described [F] is a peptide composed of any aminoacids and/or unnatural amino acids, and its length can be arbitrarilyselected depending on a physiologically active peptide to be mimicked,and the number of amino acids and/or unnatural amino acids is 1 to 20,preferably 2 to 20, more preferably 2 to 15, further preferably 3 to 10.However, the number of amino acids is not limited to this.

The sum of the numbers of amino acids or unnatural amino acids of [E],[F] and [G] is at least 3, preferably at least 4, further preferably atleast 5.

When the number of amino acids of [F] is 10 or less, the above-describedX is an alkylene chain having 1 to 8 carbon atoms, preferably amethylene chain, or a polyoxyalkylene chain having 1 to 16, preferablyhaving 1 to 8 carbon atoms, preferably a polyoxyethylene glycol chain,and Y is an alkylene chain having 1 to 8 carbon atoms, preferably amethylene chain, or a polyoxyalkylene chain having 1 to 16, preferablyhaving 1 to 8 carbon atoms, preferably a polyethylene glycol chain.

In the cross-linked peptide of the present invention, it is alsopossible to further bind any substances, for example, a low molecularweight or high molecular weight compound, a peptide, a supporting bodyor other substances to the N terminus of a peptide [E] or the C terminusof a peptide [G] or both the terminuses, and also cross-linked peptidescarrying these substances bound are included in the scope of the presentinvention. Examples thereof include, but not limited to, a low molecularweight compound or peptide showing a binding ability specific to aparticular tissue, cell or protein, a fluorescent pigment, a compoundcontaining a radioactive isotope, an intracellular migrating peptide, alow molecular weight compound or peptide having cytocidal activity suchas doxorubicin and the like, polyethylene glycol, an acyl group having 1to 18 carbon atoms, an alkyl group having 1 to 18 carbon atoms, a solidphase plate and the like.

The above-described N terminus of [E] may be protected by an aminoprotective group. Examples of the amino protective group include, butnot limited to, Fmoc, Boc, CbZ, Trityl, or an acetyl group.

The above-described C terminus of [G] may be protected by an O-esterprotective group or —NH-benzyl protective group. The O-ester protectivegroup includes, but not limited to, a —O— benzyl group, a —O-t-butylgroup, and additionally, —O-alkoxy-substituted benzyl groups describedbelow.

2. Synthesis Intermediate Compound

As the intermediate for synthesizing a cross-linked peptide of thepresent invention, the following compounds are useful.

Further, as the intermediate for synthesizing a cross-linked peptide ofthe present invention, the following compounds are useful.

X, Y, R₁, R₂, R₃ and R₄ are as described above.

[A], [B], [C] and [D] are as described above. When [A], [B], [C] or [D]is a peptide, the sum of the numbers of amino acids is at least 3,preferably at least 4, and the number of amino acids of each moiety canbe optionally selected depending on the intended cross-linked peptide.Specifically, the kind of an amino acid and/or unnatural amino acid andthe number of amino acids can be optionally selected depending on whichof [A], [B], [C] and [D] of the above-described compound corresponds toa peptide in the ring after circularization or a peptide at the Nterminus or C terminus outside of the ring after circularization.

The above-described (a) and (c) represent each independently —NH— or asingle bond, and (b) and (d) represent each independently —(C═O)— or asingle bond. When two or more (a)s, (b)s, (c)s or (d)s are contained inthe above-described formulae, each of them may be the same or different.

P₁ and P₃ represent each independently an amino protective group or ahydrogen atom, and P₂ and P₄ represent each independently an —O-esterprotective group, an —NH-benzyl protective group, an amino group or ahydroxyl group. In the above-described formula, P₁, P₂, P₃ or P₄ may bethe same or different, and when two or more P₁ s, P₂ s, P₃ s or P₄ s arecontained in the same formula, each of them may be the same ordifferent, and different is preferable.

Z represents hydrogen, an optionally substituted alkyl group having 1 to30 carbon atoms, an optionally substituted acyl group having 1 to 36carbon atoms, polyethylene glycol, a tBoc group, a Fmoc group, a Cbzgroup or a Nosyl group.

Here, the substituent is not particularly restricted providing that itcan be linked by an organic reaction, and can be optionally selecteddepending on the object of the cross-linked peptide to be synthesized.Examples thereof include, but not limited to, a protected carboxylgroup, a protected amino group, a protected thiol group, an acyl group,an alkyl group, an alkynyl group, an alkenyl group or an alkoxy group,or polyethylene glycol and the like.

Examples of the above-described amino protective group represented by P₁or P₃ include, but not limited to, Fmoc, Boc, CbZ, Trityl, an acetylgroup, and additionally, compounds working as a peptide supporting bodyin liquid phase synthesis of a peptide.

When the above-described P₁ and P₃ are both an amino protective group, acombination of protective groups capable of performing de-protectionunder different conditions is preferable. When, for example, onecomponent is a Boc group, it is preferable that the other protectivegroup is a CbZ group or a Fmoc group.

The above-described —O-ester protective group represented by P₂ or P₄includes, but not limited to, an —O-benzyl group, an —O-t-butyl group,an —O-alkoxy-substituted benzyl group, an —O-substituted methyl group, a—O-diphenylmethane derivative, and additionally, compounds working as apeptide supporting body in liquid phase synthesis of a peptide.

When the above-described P₂ and P₄ are both an —O-ester protectivegroup, a combination of protective groups capable of performingde-protection under different conditions is preferable. When, forexample, one component is an —O-t-butyl group, it is preferable that theother protective group is an —O-tetrahydropyranyl group.

Examples of the compound working as a peptide supporting body in liquidphase synthesis of a peptide (in the present specification, referredsometimes to as “Hiver”) include compounds described in JP-A No.2004-59509, compounds described in PCT international publicationWO2007/034812, compounds described in PCT international publicationWO2007/122847, compounds described in PCT international publicationWO2010/104169 and compounds described in PCT international publicationWO2010/113939. The contents described in these publications constitutepart of the present specification by incorporating herein by reference.

The preferable alkoxy-substituted benzyl group includes,

compounds having a structure described below (referred to as “Ka” insome cases in the present specification):

(wherein, R₁ and R₅ represent a hydrogen atom, and R₂, R₃ and R₄represent an alkoxyl group having 18 to 30, preferably 18 to 22 carbonatoms. In the formula, RX has a reagent active site represented by thefollowing formula.

(wherein, R₇ represents a hydrogen atom, an alkyl group having 1 to 6carbon atoms, a benzyl group or an alkoxy-substituted benzyl group, andR₆ represents a hydrogen atom, a phenyl group or an alkoxy-substitutedphenyl group.))

compounds having a structure described below (referred to as “Kb” insome cases in the present specification):

(wherein, R₂, R₄ and R₅ represent a hydrogen atom, and R₁ and R₃represent an alkoxyl group having 18 to 30, preferably 18 to 22 carbonatoms. In the formula, RY has a reagent active site represented by thefollowing formula.

(wherein, R₇ represents a hydrogen atom, an alkyl group having 1 to 6carbon atoms, a benzyl group or an alkoxy-substituted benzyl group, andR₆ represents a hydrogen atom, a phenyl group or an alkoxy-substitutedphenyl group.)), and

compounds having a structure described below (referred to as “Kc” insome cases in the present specification):

(wherein, R₁, R₃ and R₅ represent a hydrogen atom, and R₂ and R₄represent an alkoxyl group having 18 to 30, preferably 18 to 22 carbonatoms. In the formula, RZ has a reagent active site represented by thefollowing formula.

(wherein, R₇ represents a hydrogen atom, an alkyl group having 1 to 6carbon atoms, a benzyl group or an alkoxy-substituted benzyl group, andR₆ represents a hydrogen atom, a phenyl group or an alkoxy-substitutedphenyl group.)).

By using the above-described alkoxy-substituted benzyl groups Ka to Kcin combination with other amino protective groups and —O-esterprotective groups, the whole reaction for synthesizing a cross-linkedpeptide of the present invention including peptide synthesis can becarried out in a liquid phase. Ka is preferably used with designingde-protection with 50 to 100% trifluoroacetic acid, Kb is preferablyused with designing de-protection with 1 to 100% trifluoroacetic acid,and Kc is preferably used with designing de-protection with 95 to 100%trifluoroacetic acid.

It is particularly preferable to use Ka and Kb in combination or Kc andKb in combination, owing to a difference in the property thereof, forexample, because only Kb can be selectively deprotected under conditionof 1 to 5% trifluoroacetic acid.

In another preferable embodiment, it is preferable to use Kb as P₂ or P₄of one component and to use an —O-ester protective group de-protectableunder ultra-weakly acidic conditions, basic conditions and reductiveconditions as P₂ or P₄ of the other component. The protective groupcleavable under ultra-weakly acidic conditions includes an—O-tetrahydropyranyl group, the protective group cleavable under basicconditions includes an —O-fluorenylmethyl group, and the protectivegroup cleavable under reductive conditions includes an —O-benzyl group.

The cross-linked peptides (P-1) and (P-2) of the present invention canbe synthesized, for example, by reactions shown below using theabove-described intermediate compounds.

(wherein, the definitions of marks are the same as those described forthe above-described intermediates and the above-described cross-linkedpeptides.).

That is, the final cross-linked peptide (P-1) can be fabricated via anintermediate (M-1) and an intermediate (M-2) in this order. In thiscase, an endocyclic peptide sequence [F] is fabricated from peptidesequences [B] and [C] of the intermediate, and a peptide sequence [A]constitutes the N terminus and a peptide sequence [D] constitutes the Cterminus.

In contrast, the final cross-linked peptide (P-2) can be fabricated viaan intermediate (M-1) and an intermediate (M-3) in this order. In thiscase, an endocyclic peptide sequence [F] is fabricated from peptidesequences [A] and [D] of the intermediate, and an amino acid of apeptide [C] constitutes the N terminus and a peptide [B] constitutes theC terminus.

Therefore, a cross-linked peptide having a cross-linked structure at anysite in a peptide sequence can be synthesized easily. Further, since thesequence of a peptide [A], [B], [C] or [D] can be optionally selected,an endocyclic amino acid sequence and an exocyclic amino acid sequenceof the cross-linked peptide can be easily operated at will in thepresent invention. When any one or two or more of [A], [B], [C] and [D]are a single bond, also a cyclic peptide carrying no exocyclic peptidepresent or a cyclic peptide in which a peptide extends to only onedirection from the ring can be fabricated, and these are also includedin the scope of the present invention.

In the intermediate (M-1), all terminuses are protected, and theintermediate (M-2) or the intermediate (M-3) can be fabricated bydeprotecting any N terminus and any C terminus of the intermediate(M-1), and depending on the intermediate synthesis method, theintermediate (M-2) or the intermediate (M-3) can be synthesized directlynot via the intermediate (M-1).

The de-protection reaction can be carried out by a method well known tothose skilled in the art. Examples thereof include, but not limited to,de-protection with an acid such as trifluoroacetic acid, 4N—HCl/dioxaneand the like, de-protection by a catalytic hydrogenation reaction usingpalladium as a catalyst, de-protection with a base such as DBU and thelike.

A cross-linked peptide (P-1) or (P-2) of the present invention can besynthesized by performing a condensation reaction in the intermediate(M-2) or (M-3). The condensation reaction can be carried out by a methodknown to those skilled in the art, and examples thereof include, but notlimited to, a method using a carbodiimide condensation agent such asdiisopropylcarbodiimide and the like, a method using an uroniumcondensation agent such as HBTU and the like; etc.

Synthesis of a cross-linked peptide from an intermediate of anotherembodiment of the present invention will be described below.

A cross-linked peptide (P-3) of the present invention can be synthesizedby the same method as used for (M-1), as described below, using theabove-described another intermediate compound.

(wherein, the definitions of marks are as described above.).

A cross-linked peptide (P-4) of the present invention can be synthesizedby the same method as used for (M-1), as described below, using theabove-described another intermediate compound.

(wherein, the definitions of marks are as described above.).3. Synthesis of Intermediate

The intermediate compound (M-1) of the present invention can besynthesized by synthesizing a compound containing X forming part of across-linkage (here, referred to as “X side component”) and a compoundcontaining Y forming part of a cross-linkage (here, referred to as “Yside component”), separately, then, linking both the compounds.

(3-1. Synthesis of X Side Component)

An X side component can be synthesized via the following steps.

(a) a step of preparing a first component, comprising the followingsteps,

-   -   (a-1) a step of, if necessary, condensing a carboxyl protective        group with amino acids or a peptide constituting a partial        peptide sequence of the cross-linked peptide, and further, if        necessary, elongating the condensed group,    -   (a-2) a step of reacting the N terminus side of the peptide or        amino acid synthesized or the carboxyl protective group with an        amino acid derivative containing in the side chain a linker        forming part of cross-linkage of the cross-linked peptide, to        synthesize the peptide having the linker in the side chain or        the amino acid derivative having the carboxyl group protected,        and    -   (a-3) a step of, if necessary, further performing a peptide        elongation reaction to elongate the peptide.

The peptide elongation reaction can be carried out based on a knownpeptide synthesis method, and may be carried out by a solid phase methodor a liquid phase method, and from the standpoint of enhanced efficiencyof the reaction, and the like, a liquid phase synthesis method ispreferable. In the case of synthesis in a liquid phase, it is preferableto protect the C terminus of a peptide with an alkoxy-substituted benzylgroup.

Linking a compound containing a linker to the N terminus of a peptidecan be carried out by a condensation reaction known to those skilled inthe art, for example, by using the Mitsunobu reaction, however, themethod is not limited to this.

Examples of synthesis of an X side component include, but not limitedto, reactions exemplified below, and those skilled in the art can makevarious changes using known technologies.

(here, the definitions of marks are as described above, and T represents—OH, —OP₅, —NH₂, —NHP₆ or —NHNs. P₅ and P₆ represent a hydroxylprotective group and an amino protective group, respectively, known tothose skilled in the art.).

Examples of the compound providing X in synthesis of an X side componentpreferably include, but not limited to, the following compounds.

(wherein, n represents an integer of 1 to 10, preferably an integer of 1to 7, more preferably an integer of 1 to 4, and the definitions of R₁and R₄ are as described above. T represents —OH, —OP₂, —NH₂, —NHP₃ or—NHNs.).

Here, [L] is selected from the following compounds.

(wherein, n represents an integer of 1 to 10, preferably an integer of 1to 7, more preferably an integer of 1 to 4, m represents an integer of 1to 27, preferably an integer of 1 to 11, more preferably an integer of 1to 7, and l represents an integer of 1 to 24, preferably an integer of 1to 12, more preferably an integer of 1 to 8.)).

One of examples of synthesizing compounds providing various Xs, used insynthesis of an X side component, will be illustrated below.

(3-2. Synthesis of Y Side Component)

A Y side component can be synthesized via the following steps.

-   -   (b-1) a step of condensing a carboxyl protective group with        amino acids or a peptide constituting a partial peptide sequence        of the cross-linked peptide, and if necessary, elongating the        condensed group,    -   (b-2) a step of reacting the N terminus of the peptide (in this        case, it is preferable that the N terminus is, for example,        modified with Ns or modified with bromoacetyl) or the amino acid        derivative synthesized with a compound containing a linker        forming part of a cross-linkage of the cross-linked peptide, to        synthesize a peptide or an amino acid derivative having a        secondary amine at the N terminus containing the linker, and    -   (b-3) a step of, if necessary, further performing a peptide        elongation reaction to elongate the peptide.

Examples of synthesis of a Y side component include, but not limited to,reactions exemplified below, and those skilled in the art can makevarious changes using known technologies.

(The definitions are as described above.).

Another embodiment:

(The definitions are as described above.).

(3-2-1) One embodiment in which Y is the above-described compound Y1-1:

(The definitions are as described above. Here, a protective group(-(d)-P₄) attached to the C terminus side of [D] is omitted.).(3-2-2) A case in which Y is the above-described compound Y2-1:

(The definitions are as described above. Here, a protective group(-(d)-P₄) attached to the C terminus side of [D] is omitted.).(3-2-3) A case in which Y is the above-described compound Y3-1:

(The definitions are as described above, Here, a protective group(-(d)-P₄) attached to the C terminus side of [D] is omitted.).(3-2-4) A case in which Y is the above-described compound Y4-1:

(The definitions are as described above. Here, a protective group(-(d)-P₄) attached to the C terminus side of [D] is omitted.).(3-2-5) A case in which Y is the above-described compound Y5:

(The definitions are as described above. Here, a protective group(-(d)-P₄) attached to the C terminus side of [D] is omitted.).(3-3. Step of Converting Functional Group)

If necessary, it is also possible, in preparation of a first componentor a second component, to convert the functional group of a linkercontained therein into a form with which the linker can be subjected tothe subsequent cross-linkage forming reaction. As the method ofconverting a functional group, known methods can be used, and, forexample, when de-protecting a TBDPS group, it can be carried out withtetrabutylammonium fluoride, and when converting a hydroxyl group intoan aldehyde, it can be carried out by the Dess-Martin oxidationreaction.

(3-4. Synthesis of Intermediate)

Next, the intermediate (M-1) of the present invention can be synthesizedby linking an X side component and a Y side component. The condensationreaction can be carried out by a method known to those skilled in theart, and examples thereof include, but not limited to, the Mitsunobureaction, a reductive amination reaction or the Aza-Wittig reaction andthe subsequent reduction reaction. Particularly, the Mitsunobu reactionis preferable. By this, an intermediate can be synthesized having astructure in which a first component and a second component are linkedvia a secondary amine or a tertiary amine. Examples of synthesis of anintermediate include, but not limited to, reactions described below, andthose skilled in the art can make various changes using knowntechnologies.

(wherein, the definitions of marks are as described above.)

In the above-described reaction, it is necessary for the X sidecomponent and the Y side component that one of their ends pointing tothe cross-linkage center of the cross-linked peptide to be synthesizedis —NHNs and the other is —OH.

An example of synthesis of an intermediate using a reductive aminationreaction or the Aza-Wittig reaction and the subsequent reductionreaction will be illustrated below, as another embodiment of thecondensation reaction.

(wherein, the definitions of marks are as described above. Here, R₉represents a NH₂ group or a N₃ group.).

In the above-described reaction, it is necessary for the X sidecomponent and the Y side component that one of their ends pointing tothe cross-linkage center of the cross-linked peptide to be synthesizedis —NH₂ or —N₃ and the other is —CHO.

The intermediate (M-4) of the present invention can be synthesized bymutually reacting X side components, for example, using the Mitsunobureaction, but the synthesis method is not limited to this. For example,the intermediate can be synthesized by the following reaction, but thesynthesis method is not limited to the following reaction, and thoseskilled in the art can make various changes using known technologies.

(wherein, the definitions of marks are as described above, however,those represented by the same mark in the formula may be the same ordifferent.).

The intermediate (M-5) of the present invention can be synthesized bymutually reacting Y side components, for example, using the Mitsunobureaction, but the synthesis method is not limited to this. For example,the intermediate can be synthesized by the following reaction, but thesynthesis method is not limited to the following reaction, and thoseskilled in the art can make various changes using known technologies.

(wherein, the definitions of marks are as described above, however,those represented by the same mark in the formula may be the same ordifferent.).4. Method of Synthesis of Cross-Linked Peptide of the Present Invention

Synthesis of a cross-linked peptide as one embodiment of the presentinvention can be carried out via the following steps:

(a) a step of synthesizing a first component having a structuredescribed below (X side component):

(The definitions are as described above.),

(b) a step of synthesizing a second component having a structuredescribed below (Y side component):

(The definitions are as described above.),

(c) a step of reacting the above-described first component (X sidecomponent) and the above-described second component (Y side component)to synthesize an intermediate described below:

(The definitions are as described above.), and

(d) a step of synthesizing a cross-linked peptide described below fromthe above-described intermediate, by a condensation reaction:

That is, the method of synthesizing a cross-linked peptide as oneembodiment of the present invention can be carried out by a stepcontaining the following steps (a) to (g):

(a) a step of preparing a first component (component A), comprising thefollowing steps,

-   -   (a-1) a step of, if necessary, condensing a carboxyl protective        group with amino acids or a peptide constituting a partial        peptide sequence of the cross-linked peptide, and further, if        necessary, elongating the condensed group,    -   (a-2) a step of reacting the N-terminus side of the peptide or        amino acid synthesized or the carboxyl protective group with an        amino acid derivative containing in the side chain a linker        forming part of a cross-linkage of the cross-linked peptide, to        synthesize the peptide having the linker in the side chain or        the amino acid derivative having the carboxyl group protected,    -   (a-3) a step of, if necessary, further performing a peptide        elongation reaction to elongate the peptide, and    -   (a-4) a step of, if necessary (that is, when the linker end is        not reactive), converting the functional group of the linker        into a form with which the linker can be subjected to the        subsequent cross-linkage forming reaction,

(b) a step of preparing a second component (component B), comprising thefollowing steps,

-   -   (b-1) a step of condensing a carboxyl protective group with        amino acids or a peptide constituting a partial peptide sequence        of the cross-linked peptide, and if necessary, elongating the        condensed group,    -   (b-2) a step of reacting the N-terminus of the peptide or a        reaction site (for example, N-terminus) of the amino acid        derivative synthesized with a compound containing a linker        forming part of a cross-linkage of the cross-linked peptide, to        synthesize a peptide or an amino acid derivative having a        secondary amine at the N-terminus containing the linker,    -   (b-3) a step of, if necessary, further performing a peptide        elongation reaction to elongate the peptide, and    -   (b-4) a step of, if necessary (that is, when the linker end is        not reactive), converting the functional group of the linker        into a form with which the linker can be subjected to the        subsequent cross-linkage forming reaction,

(c) a step of linking the first component (A) and the second component(B) by the Mitsunobu reaction, a reductive amination reaction or theAza-Wittig reaction and the subsequent reduction reaction, to prepare anintermediate having a structure in which the first component and thesecond component are linked via a secondary amine or a tertiary amine,

(d-1) a step of, if necessary, deprotecting a protective group at thepeptide N-terminus of the first component (A) or the second component(B),

(d-2) a step of, if necessary, deprotecting a protective group at thepeptide C-terminus of the first component (A) or the second component(B),

(e) a step of condensing the peptide N- or C-terminus of the firstcomponent (A) with the peptide C- or N-terminus of the second component(B) to form a cross-linkage,

(f) a step of, if necessary, post-processing the cross-linked peptide byany method known to those skilled in the art, and

(g) a step of, if necessary, deprotecting the protective group.

Synthesis of a cross-linked peptide described below as anotherembodiment of the present invention can be carried out by a stepcomprising the following steps (a) to (g):

(a) a step of preparing a first component (component A1), comprising thefollowing steps,

-   -   (a-1) a step of, if necessary, condensing a carboxyl protective        group with amino acids or a peptide constituting a partial        peptide sequence of the cross-linked peptide, and further, if        necessary, elongating the condensed group,    -   (a-2) a step of reacting the N-terminus side of the peptide or        amino acid synthesized or the carboxyl protective group with an        amino acid derivative containing in the side chain a linker        forming part of a cross-linkage of the cross-linked peptide, to        synthesize the peptide having the linker in the side chain or        the amino acid derivative having the carboxyl group protected,    -   (a-3) a step of, if necessary, further performing a peptide        elongation reaction to elongate the peptide, and    -   (a-4) a step of, if necessary (that is, when the linker end is        not reactive), converting the functional group of the linker        into a form with which the linker can be subjected to the        subsequent cross-linkage forming reaction,

(b) a step of preparing a second component (component A2), comprisingthe following steps,

-   -   (b-1) a step of, if necessary, condensing a carboxyl protective        group with amino acids or a peptide constituting a partial        peptide sequence of the cross-linked peptide, and further, if        necessary, elongating the condensed group,    -   (b-2) a step of reacting the N-terminus side of the peptide or        amino acid synthesized or the carboxyl protective group with an        amino acid derivative containing in the side chain a linker        forming part of a cross-linkage of the cross-linked peptide, to        synthesize the peptide containing the linker in the side chain        or the amino acid derivative having the carboxyl group        protected,    -   (b-3) a step of, if necessary, further performing a peptide        elongation reaction to elongate the peptide, and    -   (b-4) a step of, if necessary (that is, when the linker end is        not reactive), converting the functional group of the linker        into a form with which the linker can be subjected to the        subsequent cross-linkage forming reaction,

(c) a step of linking the first component (A1) and the second component(A2) by the Mitsunobu reaction, a reductive amination reaction or theAza-Wittig reaction and the subsequent reduction reaction, to prepare anintermediate having a structure in which the first component (A1) andthe second component (A2) are linked via a secondary amine or a tertiaryamine,

(d-1) a step of, if necessary, deprotecting a protective group at thepeptide N-terminus of the first component (A1) or the second component(A2),

(d-2) a step of, if necessary, deprotecting a protective group at thepeptide C-terminus of the first component (A1) or the second component(A2),

(e) a step of condensing the peptide N- or C-terminus of the firstcomponent (A1) with the peptide C- or N-terminus of the second component(A2) to form a cross-linkage,

(f) a step of, if necessary, post-processing the cross-linked peptide byany method known to those skilled in the art, and

(g) a step of, if necessary, deprotecting the protective group.

Synthesis of a cross-linked peptide described below as still anotherembodiment of the present invention can be carried out by a stepcomprising the following steps (a) to (g):

(a) a step of preparing a first component (component B1), comprising thefollowing steps,

-   -   (a-1) a step of condensing a carboxyl protective group with        amino acids or a peptide constituting a partial peptide sequence        of the cross-linked peptide, and if necessary, elongating the        condensed group,    -   (a-2) a step of reacting the N-terminus of the peptide or a        reaction site (for example, N-terminus) of the amino acid        derivative synthesized with a compound containing a linker        forming part of a cross-linkage of the cross-linked peptide, to        synthesize a peptide or an amino acid derivative having a        secondary amine at the N-terminus containing the linker,    -   (a-3) a step of, if necessary, further performing a peptide        elongation reaction to elongate the peptide, and    -   (a-4) a step of, if necessary (that is, when the linker end is        not reactive), converting the functional group of the linker        into a form with which the linker can be subjected to the        subsequent cross-linkage forming reaction,

(b) a step of preparing a second component (component B2), comprisingthe following steps,

-   -   (b-1) a step of condensing a carboxyl protective group with        amino acids or a peptide constituting a partial peptide sequence        of the cross-linked peptide, and if necessary, elongating the        condensed group,    -   (b-2) a step of reacting the N-terminus of the peptide or the        amino acid derivative synthesized with a compound containing a        linker forming part of a cross-linkage of the cross-linked        peptide, to synthesize a peptide or an amino acid derivative        having a secondary amine at the N-terminus containing the        linker,    -   (b-3) a step of, if necessary, further performing a peptide        elongation reaction to elongate the peptide, and    -   (b-4) a step of, if necessary (that is, when the linker end is        not reactive), converting the functional group of the linker        into a form with which the linker can be subjected to the        subsequent cross-linkage forming reaction,

(c) a step of linking the first component (B1) and the second component(B2) by the Mitsunobu reaction, a reductive amination reaction or theAza-Wittig reaction and the subsequent reduction reaction, to prepare anintermediate having a structure in which the first component and thesecond component are linked via a secondary amine or a tertiary amine,

(d-1) a step of, if necessary, deprotecting a protective group at thepeptide N-terminus of the first component (B1) or the second component(B2),

(d-2) a step of, if necessary, deprotecting a protective group at thepeptide C-terminus of the first component (B1) or the second component(B2),

(e) a step of condensing the peptide N- or C-terminus of the firstcomponent with the peptide C- or N-terminus of the second component toform a cross-linkage,

(f) a step of, if necessary, post-processing the cross-linked peptide byany method known to those skilled in the art, and

(g) a step of, if necessary, deprotecting the protective group.

A cross-linked peptide represented by (P-1) as one embodiment of thepresent invention is used by way of example and steps thereof will beillustrated more specifically below.

4-1. Synthesis of a first component (X side component) comprises thefollowing step:

that is, a step of fabricating a first component containing a linkerintroduced, by a method comprising

(1-1) a step of preparing any amino acid in which the C-terminus isprotected with a hydrophobic carrier,

(1-2) a step of elongating a peptide using a condensation reaction and adeprotection method usually known,

(1-3) a step of introducing a linker, by protecting an amino group of anamino acid side chain acting as a linker with a nosyl group orcondensing an amino acid having a nosyl-protected amino group in theside chain or an amino acid having a protected hydroxyl group in theside chain,

(1-4) a step of, if necessary, introducing an alkylene chain acting as across-link portion onto a hydroxyl group,

(1-5) a step of, if necessary, further continuing a condensationreaction to elongate the peptide,

(1-6) a step of, if necessary, converting the functional group of thelinker into a form with which the linker can be subjected to thesubsequent cross-linkage forming reaction, and

(1-7) a step of, if necessary, introducing a —NHNs group to the linker.

By the above-described method, a first component (X side component) canbe synthesized in which the linker is an OH group or a protected OHgroup or the end is an NHNs group.

As the above-described hydrophobic carrier used for peptide synthesis ofa first component, any of carriers for solid phase synthesis andcarriers for liquid phase synthesis can be used, and preferable arehydrophobic carriers which can be used for liquid phase synthesis. Theabove-described alkoxy-substituted benzyl groups Ka, Kb and Kc areparticularly preferably used, though there is no specific restriction.

4-2. Synthesis of a second component (Y side component) comprises thefollowing step:

that is, a step of fabricating a second component containing a linkerintroduced, by a method comprising

(2-1) a step of preparing any amino acid in which the C-terminus isprotected with a hydrophobic carrier,

(2-2) a step of elongating a peptide using a condensation reaction and ade-protection method usually known,

(2-3) a step of protecting an α-amino group of an amino acid as a siteto which a linker is introduced with a nosyl group, or condensing anamino acid having a nosyl-protected α-amino group,

(2-4) a step of introducing a protected OH group using the Mitsunobureaction or other methods,

(2-5) a step of, if necessary, further continuing a condensationreaction to elongate the peptide,

(2-6) a step of, if necessary, converting the functional group of thelinker into a form with which the linker can be subjected to thesubsequent cross-linkage forming reaction, and

(2-7) a step of, if necessary, introducing a —NHNs group to the end ofthe cross-link portion.

By the above-described method, a second component (Y side component) canbe synthesized in which the linker is an OH group or a protected OHgroup or the end is an NHNs group.

As the above-described hydrophobic carrier used for peptide synthesis ofa second component, any of carriers for solid phase synthesis andcarriers for liquid phase synthesis can be used, and preferable arehydrophobic carriers which can be used for liquid phase synthesis. Theabove-described alkoxy-substituted benzyl groups Ka, Kb and Kc areparticularly preferably used, though there is no specific restriction.

Next, a first component and a second component are reacted to synthesizean intermediate. Then, the resultant intermediate is condensed tofabricate a cross-linked peptide of the present invention. In such acase, Ka, Kb and Kc are preferably used to control which of theN-terminus and the C-terminus of the first and second components shouldbe cyclized. Particularly, it is preferable that Ka or Kc is linked as aprotective group to one end and Kb is linked as a protective group tothe other end, since selective de-protection can be performed easily inthis case.

4-3. Linking of a first component and a second component can beaccomplished, for example, by combining a phosphine reagent such astriphenylphosphine and the like with a Mitsunobu reagent such as diethylazodicarboxylate and the like and performing the Mitsunobu reaction tolink a cross-link portion of the first component and a cross-linkportion of the second component. They can also be linked (cross-linked)by a reductive amination reaction or the Aza-Wittig reaction and thesubsequent reduction reaction.4-4. The step of forming a peptide bond (peptide chain) by acondensation reaction can be accomplished by subjecting any oneC-terminus and any one N-terminus of an intermediate to a condensationreaction known to those skilled in the art using a condensation agentsuch as HATU, HBTU or TBTU and the like.

The synthesis method of the present invention can include a denosylationstep of cutting a nosyl group of a synthesized cross-linked peptide,using thiophenol and DBU.

The synthesis method of the present invention can include, if necessary,a step of performing suitable chemical modification before the globaldeprotection.

The synthesis method of the present invention can include a step ofdeprotecting a side chain protective group of an amino acid of across-linked peptide by an acid or reduction treatment, therebyfabricating a naked cross-linked peptide.

The synthesis method of the present invention can include, if necessary,a step of performing suitable chemical modification after the globaldeprotection.

The method of separately synthesizing two components, for example, an Xside component and a Y side component has merits that a longer peptidecan be easily synthesized, or the kinds and lengths of endocyclic andexocyclic peptides can be freely designed and synthesized, further, alinker portion can be determined freely. Further, such a method hasmerits that a cross-link portion (portion corresponding to X and Y) canbe optionally selected and the length and the kind thereof can be easilyadjusted.

As the condensation reaction used in the present invention, any methodcan be used providing it is a method usually known in the field of aminoacid synthesis in a solid phase or a liquid phase.

As the de-protection reaction used in the present invention, any methodcan be used providing it is a method usually known or used in the fieldof amino acid synthesis, and examples thereof include a Fmoc method, aBoc method and a Z method.

The method of protecting an amino acid with nosyl used in the presentinvention can be carried out by, for example, a method of introducingnosylamide to a hydroxyl group by the Mitsunobu reaction or a method ofreacting an amino group with nosyl chloride, however, the protectingmethod is not limited to them, and it can be carried out using a methodusually known in the field of amino acid synthesis.

The nosyl-protected amino acid used in the present invention can beprepared, for example, by Fmoc-Lys-OH, however, the method is notlimited to this, and it can be prepared using a method usually known.

As the method of protecting an OH functional group of a compound capableof constituting part of a cross-linkage used in the present invention,any method can be used providing it is a method usually known in thefield of amino acid synthesis, and it can be carried out using, forexample, TMS (trimethylsilyl), TIPS (triisopropylsilyl) or TBDPS(tert-butyldiphenylsilyl), and TBDPS is preferably used. The reactioncan be carried out according to an ordinary method.

A compound constituting part of a cross-linkage in which an OHfunctional group is thus protected is introduced into a peptide.Examples of the introduction method include, but not specificallylimited to, the Mitsunobu reaction, a reductive amination reaction and acondensation reaction, and the Mitsunobu reaction is preferably used.

Here, the kind and the length of the chemical structure constitutingpart of a cross-linkage can be optionally selected and not particularlyrestricted as described previously in the present specification, andexamples thereof can include an alkylene chain, an alkyl chain, an etherchain, a thioether chain, an amide chain, a urethane chain, athiourethane chain and a polyethylene glycol chain, and preferable arean alkylene chain, an alkyl chain, an ether chain and a polyethyleneglycol chain. In the case of selection of a cross-linkage not containinga disulfide bond and an amide bond in the cross-linked structure, thereis a merit of good resistance to enzymatic degradation, for example,excellent stability against a peptidase and the like, as compared withconventional cross-linked peptides having a disulfide bond and an amidebond.

EXAMPLES

Methods of synthesizing a cross-linked peptide of the present inventionwill be illustrated using byway of example a WP9QY (W9) peptide having apeptide sequence structure containing a cross-linkage shown below, butthe present invention is not limited to them.

In the present specification and in the following examples,abbreviations shown below were used.

TABLE 1 Ac₂O: Acetic anhydride Boc: tert-Butoxycarbonyl CH₂Cl₂ (DCM):DichloromethaneCH₃CN: Acetonitrile CHCl₃: Chloroform DBU:1,8-Diazabicyclo[5.4.0]undec-7-ene DEAD: Diethyl azodicarboxylate DIPCI:N,N′-Diisopropylcarbodiimide DIPEA: N,N′-Diisopropylethylamine DMAP:4-Dimethylaminopyridine DMF: N,N-Dimethylformamide DMSO: Dimethylsulfoxide DMT-MM:4-(4,6-Dimethoxv-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride EDT:Ethanedithiol Et₃N: Triethylamine EtOAc: Ethyl acetate EtOH: EthanolFmoc: 9-Fluorenylmethoxycarbonyl HATU:O-(7-Azabenzotriazole-1-yl)1,1,3,3-tetramethyluroniumhexafluorophosphate HBTU:2-(1H-Benzotriazole-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphateHOAt: 1-Hydroxy-7-azabenzotriazole HOBt: 1-Hydroxybenzotriazole IPE:Diisopropyl Ether Ka: 3,4,5-Tri-octadesylbenzyl Kb:2,4-Di-docosyloxybenzyl MCA: Chloroacetyl MeOH: Methanol Mmt:p-Methoxytrityl Mtt: p-Methyltrityl Myr: Myristyl Ns-:2-Nitrobenzenesulfonyl NsCl: 2-Nitrobenzenesulfonyl chloride Pbf:2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl PEG:Polyethyleneglycol PhSH: Thiophenol PhSMe: Thioanisole PPh₃:Triphenylphosphine TBDPS-: tert-Butyldiphenylsilyl tBu: tert-Butyl TFA:Trifluoroacetic acid TFE: 2,2,2-Trifluoroethanol THF: TetrahydrofuranTIS: Triisopropulsilane Trt: TritylSynthesis of Cross-Linked Peptide

According to the synthesis scheme shown in FIG. 1 and FIG. 2, across-linked peptide (Bdev-7) as a peptide mimic of the W9 peptide,having —(CH₂)₄—NH—(CH₂)₄— instead of a disulfide bond as thecross-linked structure, was synthesized.

FIG. 1 is a schematic view of a synthesis route of a Y side component.FIG. 2 is a schematic view of a synthesis route in which an X sidecomponent separately synthesized is linked to a Y side component by theMitsunobu reaction to synthesize an intermediate (compound 14 in thefigure), then, a cross-linked peptide (Bdev-7) of the present inventionis synthesized by a condensation reaction. FIG. 1 and FIG. 2 onlyillustrate one route of synthesizing Bdev-7, and it is, possible to makevarious alterations and interchange synthesis routes within the scope ofthe present invention.

Each step will be illustrated in detail below.

Example 1 Synthesis of Intermediate (Compound 13) Synthesis of Compound1

2,4-didocosoxy benzyl alcohol (denoted as “KbOH”) (7.69 g, 10.1 mmol)was dissolved in CH₂Cl₂ (200 mL), and Fmoc-Gly-OH (4.52 g, 15.2 mmol,1.5 equiv), DIPCI (3147 μL, 20.2 mmol, 2.0 equiv) and DMAP (12.3 mg,0.101 mmol, 0.01 equiv) were added and the mixture was stirred at roomtemperature for 30 minutes. The precipitated material was filtrated, andthe filtrate was evaporated under reduced pressure. To the residue wasadded MeOH to find deposition of a precipitated material, which was thenfiltrated, suspended and washed with MeOH twice and suspended and washedwith CH₃CN, to obtain a compound 1 (10.1 g, 96.7%).

Synthesis of Compound 2

The compound 1 (10.1 g, 9.74 mmol) was dissolved in THF (200 mL), andpiperidine (1863 μL, 17.5 mmol, 1.8 equiv) and DBU (1863 μL, 17.5 mmol,1.8 equiv) were added and the mixture was stirred at room temperaturefor 5 minutes. Concentrated hydrochloric acid was added until pH of thereaction solution reached around 6, and the solvent was evaporated underreduced pressure. To the residue was added CH₃CN to find deposition of aprecipitated material, which was then filtrated, suspended and washedwith CH₃CN twice, to obtain a compound 2 (8.21 g, 98.7%).

Synthesis of Compound 3

The compound 2 (7.60 g, 8.96 mmol) was dissolved in CH₂Cl₂ (180 mL), andDIPEA (3589 μL, 20.6 mmol, 2.3 equiv) and NsCl (2.59 g, 11.7 mmol, 1.3equiv) were added and the mixture was stirred at room temperature. Onehour after, DIPEA (359 μL, 2.06 mmol, 0.2 equiv) was additionally addedand the mixture was stirred at room temperature for 19 minutes. To thereaction solution was added MeOH (30 mL), then, the solvent wasevaporated under reduced pressure. To the residue was added CH₃CN tofind deposition of a precipitated material, which was then filtrated,suspended and washed with CH₃CN twice, to obtain a compound 3 (8.95 g,99.9%).

Synthesis of Compound 4

The compound 3 (4.70 g, 4.71 mmol) and the compound A [1] (3.11 g, 9.46mmol, 2.0 equiv) were dissolved in THF (94 mL), PPh₃ (2.49 g, 9.50 mmol,2.0 equiv) and DEAD (4272 μL, 9.42 mmol, 2.0 equiv) were added and themixture was stirred at room temperature for 3 hours. PPh₃ (248 mg, 0.946mmol, 0.2 equiv) and DEAD (427 μL, 0.942 mmol, 0.2 equiv) were added andthe mixture was stirred for 1 hour. PPh₃ (245 mg, 0.934 mmol, 0.2 equiv)and DEAD (427 μL, 0.942 mmol, 0.2 equiv) were added and the mixture wasstirred for 1 hour and 15 minutes, then, the solvent was evaporatedunder reduced pressure. To the residue was added CH₃CN to finddeposition of a precipitated material, which was then filtrated,suspended and washed with CH₃CN twice, to obtain a compound 4 (6.12 g,99.2%).

Synthesis of Compound 5

The compound 4 (5.65 g, 4.31 mmol) was dissolved in THF (40 mL), PhSH(1328 μL, 12.9 mmol, 3.0 equiv) and DBU (1934 μL, 12.9 mmol, 3.0 equiv)were added and the mixture was stirred at room temperature for 1 hourand 30 minutes. To the reaction solution was added CH₃CN (200 mL) tofind deposition of a precipitated material, which was then filtrated,suspended and washed with CH₃CN twice, to obtain a compound 5 (4.77 g,98.4%).

Synthesis of Compound 6

The compound 5 (7.49 g, 6.65 mmol) was dissolved in THF (70 mL), andFmoc-Leu-OH (3.53 g, 9.98 mmol, 1.5 equiv), HOAt (2.27 g, 16.7 mmol, 2.5equiv), HATU (6.32 g, 16.6 mmol, 2.5 equiv) and DIPEA (5800 μL, 33.3mmol, 5.0 equiv) were added and the mixture was stirred at roomtemperature for 6 minutes. DIPEA (580 μL, 3.33 mmol, 0.5 equiv) wasadded and the mixture was stirred for 8 minutes. DIPEA (580 μL, 3.33mmol, 0.5 equiv) was added and the mixture was stirred for 1 hour. Theprecipitated material was filtrated, and the filtrate was evaporatedunder reduced pressure. The same post treatment as in synthesis of thecompound 4 was conducted to obtain a residue, which was then subjectedto silica gel column chromatography (n-hexane/EtOAc=100:0-85:15), toobtain a compound 6 (9.10 g, 93.7%).

Synthesis of Compound 7

The compound 6 (1.05 g, 0.721 mmol) was dissolved in CH₂Cl₂ (36 mL), andTFE (3.6 mL) and TFA (360 μl) were added and the mixture was stirred atroom temperature for 40 minutes. The precipitated material wasfiltrated, and the filtrate was evaporated under reduced pressure. Tothe residue was added CH₂Cl₂ and the mixture was washed with water threetimes, the organic layer was washed with saturated saline, dried overanhydrous MgSO₄, filtrated and the filtrate was evaporated under reducedpressure. The residue was subjected to silica gel column chromatography(CH₂Cl₂/MeOH, 100:0-90:10), to obtain a compound 7 (478 mg, 92.2%).

Synthesis of Compound 8

The compound 7 (4.08 g, 5.66 mmol, 1.2 equiv) was dissolved in THF (70mL), and the compound 29 (synthesis method is described later) (5.52 g,4.72 mmol), DIPCI (1104 μL, 7.09 mmol, 1.5 equiv), HOAt (963 mg, 7.08mmol, 1.5 equiv) and DIPEA (4940 μL, 28.4 mmol, 6.0 equiv) were addedand the mixture was stirred at room temperature for 3 hours. HOAt (325mg, 2.39 mmol, 0.5 equiv) and DIPCI (368 μL, 2.36 mmol, 0.5 equiv) wereadded and the mixture was stirred for 2 hours. The solvent wasevaporated under reduced pressure, then, the same post treatment as insynthesis of the compound 1 was conducted, to obtain a compound 8 (8.54g, 98.5%).

Synthesis of Compound 9

The compound 8 (8.54 g, 4.65 mmol) was dissolved in THF (93 mL), andpiperidine (891 μL, 8.37 mmol, 1.8 equiv) and DBU (904 μL, 6.04 mmol,1.3 equiv) were added and the mixture was stirred at room temperaturefor 5 minutes. The same post treatment as in synthesis of the compound 2was conducted, to obtain a de-Fmoc form (8.31 g).

The de-Fmoc form (8.31 g) was dissolved in THF (93 mL), andFmoc-Tyr(tBu)-OH (3.21 g, 6.99 mmol, 1.5 equiv), HOAt (1.59 g, 11.7mmol, 2.5 equiv), HATU (4.42 g, 11.6 mmol, 2.5 equiv) and DIPEA (4860μL, 27.9 mmol, 6.0 equiv) were added and the mixture was stirred at roomtemperature for 40 minutes. The same post treatment as in synthesis ofthe compound 6 was conducted, to obtain a compound 9 (9.05 g, 94.7%).

Synthesis of Compound 10

The compound 9 (4.17 g, 2.03 mmol) was dissolved in THF (41 mL), andpiperidine (389 μL, 3.65 mmol, 1.8 equiv) and DBU (395 μL, 2.63 mmol,1.3 equiv) were added and the mixture was stirred at room temperaturefor 5 minutes. The same post treatment as in synthesis of the compound 2was conducted, to obtain a de-Fmoc form (3.92 g).

The de-Fmoc form (3.92 g) was dissolved in THF (41 mL), andFmoc-Gln(Trt)-OH (1.86 g, 3.04 mmol, 1.5 equiv), HATU (1.93 g, 5.07mmol, 2.5 equiv), HOAt (691 mg, 5.07 mmol, 2.5 equiv) and DIPEA (2120μL, 12.2 mmol, 6.0 equiv) were added and the mixture was stirred at roomtemperature for 30 minutes. The same post treatment as in synthesis ofthe compound 4 was conducted, to obtain a compound 10 (4.83 g, 98.0%).

Synthesis of Compound 11

The compound 10 (4.83 g, 1.99 mmol) was dissolved in THF (40 mL), andpiperidine (382 μL, 3.59 mmol, 1.8 equiv) and DBU (388 μL, 2.59 mmol,1.3 equiv) were added and the mixture was stirred at room temperaturefor 5 minutes. The same post treatment as in synthesis of the compound 2was conducted, to obtain a de-Fmoc form (4.63 g).

The de-Fmoc form (4.63 g) was dissolved in THF (40 mL), andFmoc-Ser(tBu)-OH (1.15 g, 2.99 mmol, 1.5 equiv), HATU (1.89 g, 4.98mmol, 2.5 equiv), HOAt (676 mg, 4.98 mmol, 2.5 equiv) and DIPEA (2082μL, 11.9 mmol, 6.0 equiv) were added and the mixture was stirred at roomtemperature for 45 minutes. The same post treatment as in synthesis ofthe compound 4 was conducted, to obtain a compound 11 (5.49 g).

Synthesis of Compound 12

The compound 11 (5.49 g) was dissolved in THF (40 mL), and piperidine(382 μL, 3.59 mmol, 1.8 equiv) and DBU (388 μL, 2.59 mmol, 1.3 equiv)were added and the mixture was stirred at room temperature for 5minutes. The same post treatment as in synthesis of the compound 2 wasconducted, to obtain a de-Fmoc form (4.65 g, 98.0% from compound 10).

The de-Fmoc form (4.65 g, 1.95 mmol) was dissolved in THF (39 mL), andFmoc-Trp(Boc)-OH (1.54 g, 2.92 mmol, 1.5 equiv), HOAt (398 mg, 2.92mmol, 1.5 equiv), HATU (1.11 g, 2.92 mmol, 1.5 equiv) and DIPEA (1698μL, 9.75 mmol, 5.0 equiv) were added and the mixture was stirred at roomtemperature for 70 minutes. The same post treatment as in synthesis ofthe compound 4 was conducted, to obtain a compound 12 (5.32 g, 95.4%).

Synthesis of Compound 13

The compound 12 (5.32 g, 1.86 mmol) was dissolved in THF (37.2 mL), andpiperidine (372 μL, 3.50 mmol, 1.9 equiv) and DBU (372 μL, 2.49 mmol,1.3 equiv) were added and the mixture was stirred at room temperaturefor 5 minutes. The same post treatment as in synthesis of the compound 2was conducted, to obtain a de-Fmoc form (4.96 g).

The de-Fmoc form (4.96 g) was dissolved in THF (12.6 mL), and TBAF (1.0M solution in THF, 6.0 mL, 6.00 mmol, 3.2 equiv) was added and themixture was stirred at room temperature for 19 hours. The solvent wasevaporated under reduced pressure, then, to the residue was added CH₂Cl₂and the mixture was washed with 1 N HCl, water and saturated saline, theorganic layer was dried over anhydrous MgSO₄, filtrated, then, thefiltrate was evaporated under reduced pressure. The residue wassubjected to silica gel column chromatography (CH₂Cl₂/THF, 100:0-75:25),to obtain a compound 13 (2.02 g, 45.3%).

The compound 13 is a component in which [C] is Trp-Ser-Gln-Tyr-Leu and[D] is Tyr.

Synthesis of Compound 29

3,4,5-trioctadecyloxybenzyl alcohol (denoted as “KaOH”) (20.1 g, 22.0mmol) was dissolved in CH₂Cl₂ (220 mL), and Fmoc-Tyr(tBu)-OH (15.2 g,33.0 mmol, 1.5 equiv), DIPCI (6856 μL, 44.0 mmol, 2.0 equiv) and DMAP(26.9 mg, 0.220 mmol, 0.01 equiv) were added and the mixture was stirredat room temperature for 35 minutes. The same post treatment as insynthesis of the compound 1 was conducted, to obtain a compound 28 (29.9g, q. y.).

The compound 28 (9.58 g, 7.07 mmol) was dissolved in THF (141 mL), andpiperidine (1260 μL, 12.7 mmol, 1.8 equiv) and DBU (1374 μL, 9.19 mmol,1.3 equiv) were added and the mixture was stirred at room temperaturefor 5 minutes. After confirmation of disappearance of raw materials byTLC, the same post treatment as described above (synthesis of compound2) was conducted, to obtain a compound 29 (8.28 g, q. y.).

Example 2 Synthesis of Intermediate (Compound 38) Synthesis of FirstComponent

The compound 38 as a first component (X side component) was synthesizedby the following step.

Synthesis of Compound 30

3,4,5-trioctadecyloxybenzyl alcohol (hereinafter, denoted as “KaOH”)(3.00 g, 3.28 mmol) was dissolved in CH₂Cl₂ (32.8 mL), andFmoc-Lys(Mtt)-OH (3.08 g, 4.93 mmol, 1.5 equiv), DIPCI (1023 μL, 6.57mmol, 2.0 equiv) and DMAP (4.0 mg, 0.0328 mmol, 0.01 equiv) were addedand the mixture was stirred at room temperature for 42 minutes. The samepost treatment as in synthesis of the compound 1 was conducted, toobtain a compound 30 (5.32 g).

Synthesis of Compound 31

The compound 30 (5.32 g) was dissolved in CH₂Cl₂ (32.8 mL), and TIS(3284 μL) and TFA (985.2 μL) were added and the mixture was stirred atroom temperature for 12 minutes. TIS (3284 μl) was additionally addedand the mixture was further stirred for 53 minutes. TFA (985.2 μL) wasadditionally added and the mixture was further stirred for 16 minutes.The same post treatment as in synthesis of the compound 4 was conducted,to obtain a compound 31 (4.48 g).

Synthesis of Compound 32

The compound 31 (4.48 g) was dissolved in CH₂Cl₂ (65.7 mL), and DIPEA(1258 μL, 7.22 mmol, 2.2 equiv) and NsCl (873 mg, 3.94 mmol, 1.2 equiv)were added and the mixture was stirred at room temperature for 37minutes. The same post treatment as in synthesis of the compound 3 wasconducted, to obtain a compound 32 (4.75 g, q. y.).

Synthesis of Compound 33

The compound 32 (4.38 g, 3.02 mmol) was dissolved in CH₂Cl₂ (27 mL), andTIS (6.0 mL) and TFA (27 mL) were added and the mixture was stirred atroom temperature for 2 hours. The solvent was evaporated under reducedpressure, to the residue was added CH₂Cl₂ to give a diluted solution,which was then washed with water three times and saturated saline, andthe organic layer was dried over anhydrous MgSO₄, filtrated and thefiltrate was evaporated under reduced pressure. The residue wassubjected to silica gel column chromatography (CH₂Cl₂/MeOH,100:0-85:15), to obtain a compound 33 (1.42 g, 84.8%).

Synthesis of Compound 34

2,4-didocosoxy benzyl alcohol (hereinafter, denoted as “KbOH”) (2.09 g,2.76 mmol) was dissolved in CH₂Cl₂ (27.6 mL), and the compound 33 (2.29g, 4.14 mmol, 1.5 equiv), DIPCI (859 μL, 5.51 mmol, 2.0 equiv) and DMAP(3.4 mg, 0.0278 mmol, 0.01 equiv) were added and the mixture was stirredat room temperature for 45 minutes. The same post treatment as for thecompound 1 was conducted, to obtain a compound 34 (4.13 g, q. y.).

Synthesis of Compound 35

The compound 34 (4.13) was dissolved in THF (55.1 mL), and piperidine(491 μL) and DBU (536 μL) were added and the mixture was stirred at roomtemperature for 5 minutes. After confirmation of disappearance ofstarting materials by TLC, the same post treatment as in synthesis ofthe compound 2 was conducted, to obtain a compound 35 (3.00 g, 98.2%).

Synthesis of Compound 36

The compound 35 (332 mg, 0.300 mmol) was dissolved in THF (6 mL), andFmoc-Tyr(tBu)-OH (207 mg, 0.450 mmol, 1.5 equiv), HOAt (61.3 mg, 0.450mmol, 1.5 equiv), HATU (171 mg, 0.450 mmol, 1.5 equiv) and DIPEA (261μL, 1.50 mmol, 5.0 equiv) were added and the mixture was stirred at roomtemperature for 53 minutes. The same post treatment as in synthesis ofthe compound 4 was conducted, to obtain a compound 36 (441 mg, 97.3%).

Synthesis of Compound 37

The compound 36 (2.73 g, 1.81 mmol) was dissolved in THF (36.1 mL), andpiperidine (322 μL) and DBU (351 μL) were added and the mixture wasstirred at room temperature for 5 minutes. After confirmation ofdisappearance of starting materials by TLC, the same post treatment asin synthesis of the compound 2 was conducted, to obtain a compound 37(2.25 g, 94.1%).

Synthesis of Compound 38

The compound 37 (266 mg, 0.201 mmol) was dissolved in THF (4 mL), andmyristic acid (68.9 mg, 0.302 mmol, 1.5 equiv), HOAt (41.4 mg, 0.304mmol, 1.5 equiv), HATU (114 mg, 0.301 mmol, 1.5 equiv) and DIPEA (174μL, 0.999 mmol, 5.0 equiv) were added and the mixture was stirred atroom temperature for 1 hour and 50 minutes. The same post treatment asin synthesis of the compound 4 was conducted, to obtain a compound 38(289 mg, 95.5%).

The compound 38 is a component in which [A] is Tyr and [B] is a singlebond.

Synthesis of Intermediate Synthesis of Compound 14

The compound 13 (265 mg, 0.111 mmol), the compound 38 (289 mg, 0.192mmol, 1.7 equiv) and PPh₃ (118 mg, 0.450 mmol, 4.1 equiv) were dissolvedin THF (11 mL), and DEAD (201 μL, 0.443 mmol, 4.0 equiv) was added andthe mixture was stirred at room temperature for 61 minutes. PPh₃ (121mg, 0.461 mmol, 4.2 equiv) and DEAD (201 μL, 0.443 mmol, 4.0 equiv) wereadded and the mixture was stirred for 59 minutes. The same posttreatment as in synthesis of the compound 4 was conducted to obtain aresidue, which was then subjected to silica gel column chromatography(CH₂CH₂/THF, 100:0-88:12), to obtain a Mitsunobu reaction product (132mg, 30.6%).

The Mitsunobu reaction product (132 mg, 0.0340 mmol) was dissolved inCH₂Cl₂ (3.4 mL), and TFE (340 μL) and TFA (34.0 μL) were added and themixture was stirred at room temperature for 70 minutes. The precipitatedmaterial was filtrated through Celite, and the solvent was evaporatedunder reduced pressure. The same post treatment as in synthesis of thecompound 4 was conducted to obtain a residue, which was then subjectedto silica gel column chromatography (CHCl₃/MeOH, 100:0-95:5), to obtaina compound 14 (75.1 mg, 70.3%) as an intermediate of the presentinvention.

Example 3 Synthesis of Cross-Linked Peptide (Bdev-7) Synthesis ofCompound 15

The compound 14 (75.1 mg, 0.0239 mmol), HOAt (4.2 mg, 0.0309 mmol, 1.3equiv) and HATU (11.2 mg, 0.0295 mmol, 1.2 equiv) were dissolved in THF(4780 μL), and DIPEA (20.8 μL, 0.119 mmol, 5.0 equiv) was added and themixture was stirred at room temperature for 17 hours and 40 minutes. Thesame post treatment as in synthesis of the compound 4 was conducted, toobtain a compound 15 (64.5 mg, 86.6%).

Synthesis of Compound 16

The compound 15 (64.5 mg, 0.0207 mmol) was dissolved in THF (414 μL),and PhSH (6.38 μL, 0.0621 mmol, 3.0 equiv) and DBU (9.29 μL, 0.0621mmol, 3.0 equiv) were added and the mixture was stirred at roomtemperature for 1 hour and 38 minutes. PhSH (6.38 μL, 0.0621 mmol, 3.0equiv) and DBU (9.29 μL, 0.0621 mmol, 3.0 equiv) were added and themixture was stirred at room temperature for 3 hours and 29 minutes. Tothe reaction solution was added concentrated hydrochloric acid (10 μL)and the solvent was evaporated under reduced pressure. The same posttreatment as in synthesis of the compound 4 was conducted, to obtain acompound 16 (55.6 mg, 91.3%).

Synthesis of Cross-Linked Peptide (Bdev-7)

To the compound 16 (21.8 mg, 0.0743 mmol) was added a solution (743 μL)of TFA/TIS/H₂O=95:5:5 and the mixture was stirred at room temperaturefor 3 hours. The precipitated material was filtrated, and the filtratewas evaporated under reduced pressure. To the residue was added IPE tofind deposition of a precipitated material, which was centrifugallyseparated to give a residue, and the residue was purified by HPLC, toobtain a cross-linked peptide of the present invention (Bdev-7) (2.8 mg,2.6%). HRMS m/z [M+H]⁺: calcd for C₇₈H₁₁₁N₁₂O₁₆: 1471.8241. found1472.1734.

Cross-linked peptides as other W9 peptide mimics having the followingpeptide sequence structures were synthesized. The structure patterns andZ₁ to Z₃ substitution patterns of the compounds synthesized are asdescribed in Table 2 below.

TABLE 2 compound structure substitution pattern number pattern Z₁ Z₃ Z₂Bdev-2 P-1 H Ac OH Bdev-3 P-1 Ac PEG₂₀₀₀ OH Bdev-4 P-1 H Myr OH Bdev-5P-1 Ac Ac OH Bdev-6 P-1 Ac H OH Bdev-7*¹ P-1 Myr H OH Bdev-8 P-1 H H OHBdev-10 P-1 PEG₂₀₀₀ Ac OH Bdev-12 P-1 Ac Myr OH Bdev-13 P-1 Ac AcNH-PEG₂₀₀₀ Bdev-14 P-1 H PEG₂₀₀₀ OH Bdev-19 P-2 H Ac OH Bdev-20 P-2 HPEG₂₀₀₀ OH Bdev-21 P-3 H Ac OH Bdev-25 P-4 H Ac OH Bdev-27 P-1 H EtC(O)OH Bdev-28 P-1 H n-BuC(O) OH Bdev-29 P-1 H mPEG3 OH Bdev-30 P-1 H mPEG7OH *¹compound synthesized in Example 2

Example 4 Synthesis of Intermediate (Compound 19) for Synthesis ofBdev-3, -5, -6, -12 and -13

Synthesis of an intermediate for synthesis of Bdev-3, -5, -6, -12 and-13 was carried out as described below.

Synthesis of Compound 39

The compound 37 (746 mg, 0.562 mmol) was dissolved in CH₂Cl₂ (11 mL),and Et₃N (160 μL, 1.14 mmol, 2.0 equiv) and Ac₂O (100 μL, 1.06 mmol, 1.9equiv) were added and the mixture was stirred at room temperature for 52minutes. The same post treatment as described above (synthesis ofcompound 3) was conducted, to obtain a compound 39 (698 mg, 0.524 mmol,93.2%).

Synthesis of Compound 40

The compound 35 (775 mg, 0.700 mmol) was dissolved in THF (14 mL), andBoc-Tyr(tBu)-OH (354 mg, 1.05 mmol, 1.5 equiv), HOAt (143 mg, 1.05 mmol,1.5 equiv), HATU (399 mg, 1.05 mmol, 1.5 equiv) and DIPEA (610 μL, 3.50mmol, 5.0 equiv) were added and the mixture was stirred at roomtemperature for 42 minutes. The same post treatment as in synthesis ofthe compound 4 was conducted, to obtain a compound 40 (935 mg, 96.1%).

Synthesis of Compound 17

The compound 13 (944 mg, 0.394 mmol), the compound 39 (635 mg, 0.477mmol, 1.2 equiv) and PPh₃ (211 mg, 0.804 mmol, 2.0 equiv) were dissolvedin THF (40 mL), and DEAD (357 μL, 0.787 mmol, 2.0 equiv) was added andthe mixture was stirred at room temperature for 3 hours and 7 minutes.PPh₃ (208 mg, 0.793 mmol, 2.0 equiv) was added and the mixture wasstirred for 33 minutes. DEAD (357 μL, 0.787 mmol, 2.0 equiv) was addedand the mixture was stirred or 1 hour and 50 minutes. The same posttreatment as in synthesis of the compound 4 was conducted to obtain aresidue (1.573 g), which was used in the subsequent reaction.

The residue (1.573 g) was dissolved in CH₂Cl₂ (47.7 mL), and TFE (4770μL) and TFA (477 μL) were added and the mixture was stirred at roomtemperature for 1 hour. The precipitated material was filtrated throughCelite, and to the filtrate was added DIPEA (1050 μL), then, the solventwas evaporated under reduced pressure. The same post treatment as insynthesis of the compound 4 was conducted to obtain a residue, which wasthen subjected to silica gel column chromatography (CHCl₃/MeOH,100:0-90:10 and CHCl₃/EtOH, 100:0-93:7), to obtain a compound 17 (651mg, 55.7%) as an intermediate of the present invention.

Synthesis of Compound 18

The compound 17 (485 mg, 0.163 mmol), HOAt (26.8 mg, 0.197 mmol, 1.2equiv) and HATU (74.8 mg, 0.197 mmol, 1.2 equiv) were dissolved in THF(32.6 mL), and DIPEA (142 μL, 0.815 mmol, 5.0 equiv) was added and themixture was stirred at room temperature for 3 hours and 30 minutes. Thesame post treatment as in synthesis of the compound 4 was conducted, toobtain a compound 18 (462 mg, 96.3%).

Synthesis of Compound 19

The compound 40 (462 mg, 0.157 mmol) was dissolved in THF (1560 μL), andPhSH (16.0 μL, 0.156 mmol, 1.0 equiv) and DBU (70.0 μL, 0.468 mmol, 3.0equiv) were added and the mixture was stirred at room temperature for 50minutes. PhSH (32.0 μL, 0.312 mmol, 2.0 equiv) was added and the mixturewas stirred for 1 hour and 11 minutes. PhSH (48.0 μL, 0.467 mmol, 3.0equiv) and DBU (70.0 μL, 0.468 mmol, 3.0 equiv) were added and themixture was stirred at room temperature for 50 minutes. To the reactionsolution was added concentrated hydrochloric acid (78.0 μL) and thesolvent was evaporated under reduced pressure. The same post treatmentas in synthesis of the compound 16 was conducted, to obtain a compound19 (416 mg, 96.2%).

Example 5 Synthesis of Bdev-3, -5, -6, -12 and -13 (1) Synthesis ofBdev-6

Bdev-6 was synthesized as described below.

To the compound 19 (48.2 mg, 0.0174 mmol) was added a solution (1740 μL)of TFA/TIS/H₂O=95:5:5 and the mixture was stirred at room temperaturefor 3 hours. The same post treatment and HPLC purification as insynthesis of Bdev-7 were conducted, to obtain Bdev-6 (5.3 mg, 23.4%).HRMS m/z [M+H]⁺: calcd for C₆₆H₈₇N₁₂O₁₆: 1303.6363. found 1303.9331.

(2) Synthesis of Bdev-12

Bdev-12 was synthesized as described below.

Synthesis of Compound 20

The compound 19 (149 mg, 0.0540 mmol), myristic acid (18.7 mg, 0.0819mmol, 1.5 equiv), HOAt (11.1 mg, 0.0816 mmol, 1.5 equiv) and HATU (31.0mg, 0.0815 mmol, 1.5 equiv) were dissolved in THF (1080 μL), and DIPEA(47.0 μL, 0.270 mmol, 5.0 equiv) was added and the mixture was stirredat room temperature for 3 hours. The same post treatment as in synthesisof the compound 4 was conducted, to obtain a compound 20 (147 mg,91.5%).

Synthesis of Bdev-12

To the compound 20 (147 mg, 0.0494 mmol) was added a solution (5 mL) ofTFA/TIS/H₂O=95:5:5 and the mixture was stirred at room temperature for 3hours. The same post treatment and HPLC purification as in synthesis ofBdev-7 were conducted, to obtain Bdev-12 (28.0 mg, 37.4%). HRMS m/z[M+H]⁺: calcd for C₈₀H₁₁₃N₁₂O₁₇: 1513.8347. found 1514.2531.

(3) Synthesis of Bdev-3

Bdev-3 was synthesized as described below.

Synthesis of Compound 21

The compound 19 (111 mg, 0.0400 mmol), MeO-PEG-CO₂H (121 mg, 0.0600mmol, 1.5 equiv), HOAt (6.5 mg, 0.0480 mmol, 1.2 equiv) and HATU (18.3mg, 0.0480 mmol, 1.2 equiv) were dissolved in THF (2 mL), and DIPEA(34.8 μL, 0.200 mmol, 5.0 equiv) was added and the mixture was stirredat room temperature for 53 minutes. DIPEA (34.8 μL, 0.200 mmol, 5.0equiv) was added and the mixture was stirred for 56 minutes.MeO-PEG-CO₂H (121 mg, 0.0600 mmol, 1.5 equiv), HATU (22.8 mg, 0.0600mmol, 1.5 equiv) and DIPEA (17.4 mL, 0.0999 mmol, 2.5 equiv) were addedand the mixture was stirred for 2 hours and 9 minutes. The solvent wasevaporated under reduced pressure, and the residue was subjected tosilica gel column chromatography (CH₂Cl₂/MeOH, 100:0-90:10), to obtain acompound 21 (313 mg) as a crude product.

Synthesis of Bdev-3

To the compound 21 (147 mg, 0.0494 mmol) was added a solution (5 mL) ofTFA/TIS/H₂O=95:5:5 and the mixture was stirred at room temperature for 3hours. The same post treatment and HPLC purification as in synthesis ofBdev-7 were conducted, to obtain Bdev-3 (28.0 mg, 37.4%). The structureof the targeted substance was identified based on detection ofmono-valent ions at 44-amu interval around m/z 3140 and divalent ions at22-amu interval around m/z 1640 by MS measurement.

(4) Synthesis of Bdev-5

Bdev-5 was synthesized as described below.

Synthesis of Compound 22

The compound 19 (161 mg, 0.0580 mmol) was dissolved in CH₂Cl₂ (1160 μL),and Et₃N (15.8 μL, 0.112 mmol, 1.9 equiv) and Ac₂O (11.0 μL, 0.116 mmol,2.0 equiv) were added and the mixture was stirred at room temperaturefor 40 minutes. The same post treatment as in synthesis of the compound4 was conducted, to obtain a compound 22 (143 mg, 87.6%).

Synthesis of Bdev-5

To the compound 22 (143 mg, 0.0508 mmol) was added a solution (5 mL) ofTFA/TIS/H₂O=95:5:5 and the mixture was stirred at room temperature for 3hours. The same post treatment and HPLC purification as in synthesis ofBdev-7 were conducted, to obtain Bdev-5 (25.6 mg, 37.4%). HRMS m/z[M+H]⁺: calcd for C₆₈H₈₈N₂O₁₇: 1345.6499. found 1345.5516.

(5) Synthesis of Bdev-13

Bdev-13 was synthesized as described below.

A mixture of Bdev-5 (10.1 mg, 0.00750 mmol, 1.5 equiv), MeO-PEG-NH₂ (MW:2000 Da, manufactured by Iris Biotech) (10.4 mg, 0.00516 mmol) andDMT-MM (1.4 mg, 0.00506 mmol, 1.0 equiv) in THF (1100 μL) and MeOH (900μL) was stirred at room temperature for 7 hours and 35 minutes. Thesolvent was evaporated under reduced pressure, then, to the residue wereadded PEG-NH₂ (10.4 mg, 0.00516 mmol), DMT-MM (5.7 mg, 0.0206 mmol) andDMF (1 mL) and the mixture was stirred at room temperature for 4 hoursand 38 minutes. DMT-MM (14.2 mg, 0.0513 mmol) was added and the mixturewas stirred for 1 hour and 20 minutes. Purification was performed byHPLC, to obtain Bdev-13 (3.6 mg, 14.5%). The structure of the targetedsubstance was identified based on detection of a group of divalent ionsat 22-amu interval around m/z 1700, a group of tri-valent ions at 15-amuinterval around m/z 1150 and a group of tetra-valent ions at 11-amuinterval around m/z 900 by MS measurement.

Example 6 Synthesis of Intermediate for Synthesis of Bdev-2, -4, -8, and-10

Synthesis of an intermediate for synthesis of Bdev-2, -4, -8, and -10was carried out as described below.

Synthesis of Compound 23

The compound 13 (890 mg, 0.372 mmol), the compound 40 (776 mg, 0.559mmol, 1.5 equiv) and PPh₃ (147 mg, 0.559 mmol, 1.5 equiv) were dissolvedin THF (8 mL), and DEAD (253 μL, 0.558 mmol, 1.5 equiv) was added andthe mixture was stirred at room temperature for 54 minutes. PPh₃ (148mg, 0.564 mmol, 1.5 equiv) was added and the mixture was stirred for 20minutes. DEAD (84.4 μL, 0.186 mmol, 0.5 equiv) was added and the mixturewas stirred for 2 hours and 20 minutes. PPh₃ (147 mg, 0.560 mmol, 1.5equiv) was added and the mixture was stirred for 14 minutes. DEAD (33.7μL, 0.0743 mmol, 0.2 equiv) was added and the mixture was stirred for 34minutes. PPh₃ (44.8 mg, 0.171 mmol, 0.5 equiv) was added and the mixturewas stirred for 2 minutes. DEAD (33.7 μL, 0.0743 mmol, 0.2 equiv) wasadded and the mixture was stirred for 3 hours and 15 minutes. DEAD (33.7μL, 0.0743 mmol, 0.2 equiv) was added and the mixture was stirred for 21minutes. The same post treatment as in synthesis of the compound 4 wasconducted to obtain a residue, which was then subjected to silica gelcolumn chromatography (CH₂CH₂/THF, 100:0-90:10), to obtain a Mitsunobureaction product (1.05 g, 75.0%).

The Mitsunobu reaction product (637 mg, 0.169 mmol) was dissolved inCH₂Cl₂ (16.9 mL), and TFE (1690 μL) and TFA (169 μL) were added and themixture was stirred at room temperature for 40 minutes. The precipitatedmaterial was filtrated through Celite, and the solvent was evaporatedunder reduced pressure. The same post treatment as in synthesis of thecompound 4 was conducted to obtain a residue, which was then subjectedto silica gel column chromatography (CHCl₃/EtOH, 100:0-95:5), to obtaina compound 23 (294 mg, 58.1%).

Synthesis of Compound 24

The compound 23 (294 mg, 0.0970 mmol), HOAt (15.8 mg, 0.116 mmol, 1.2equiv) and HATU (44.7 mg, 0.118 mmol, 1.2 equiv) were dissolved in THF(19.4 mL), and DIPEA (84.5 μL, 0.485 mmol, 5.0 equiv) was added and themixture was stirred at room temperature for 3 hours and 10 minutes. Thesame post treatment as in synthesis of the compound 4 was conducted, toobtain a compound 24 (284 mg, 97.2%).

Synthesis of Compound 25

The compound 24 (284 mg, 0.0943 mmol) was dissolved in THF (943 μL), andPhSH (29.0 μL, 0.282 mmol, 3.0 equiv) and DBU (42.3 μL, 0.283 mmol, 3.0equiv) were added and the mixture was stirred at room temperature for 2hours and 30 minutes. The same post treatment as in synthesis of thecompound 16 was conducted, to obtain a compound 25 (255 mg, 95.8%) as anintermediate for synthesis of Bdev-2, -4, -8 and -10.

Example 7 Synthesis of Bdev-2, -4, -8 and -10 (1) Synthesis of Bdev-8

Synthesis of Bdev-8 was carried out as described below.

To the compound 25 (55.1 mg, 0.0181 mmol) was added a solution (1810 μL)of TFA/TIS/H₂O=95:5:5 and the mixture was stirred at room temperaturefor 3 hours. The same post treatment and HPLC purification as insynthesis of Bdev-7 were conducted, to obtain Bdev-8 (12.0 mg, 52.6%).HRMS m/z [M+H]⁺: calcd for C₆₄H₈₅H₁₂O₁₅: 1261.6257. found 1261.4668.

(2) Synthesis of Bdev-4 Synthesis of Compound 26

The compound 25 (62.6 mg, 0.0222 mmol), myristic acid (7.7 mg, 0.0337mmol, 1.5 equiv), HOAt (4.5 mg, 0.0331 mmol, 1.5 equiv) and HATU (12.8mg, 0.0337 mmol, 1.5 equiv) were dissolved in THF (444 μL), and DIPEA(19.3 μL, 0.111 mmol, 5.0 equiv) was added and the mixture was stirredat room temperature for 3 hours. The same post treatment as in synthesisof the compound 4 was conducted, to obtain a compound 26 (56.9 mg,84.7%).

Synthesis of Bdev-4

To the compound 26 (56.9 mg, 0.0188 mmol) was added a solution (1514 μL)of TFA/TIS/H₂O=95:5:5 and the mixture was stirred at room temperaturefor 5 minutes. TIS (46.9 mL) was added and the mixture was stirred for 3hours and 55 minutes. The same post treatment as in synthesis of Bdev-7was conducted, to obtain Bdev-4 (4.0 mg, 14.5%). HRMS m/z [M+H]⁺: calcdfor C₇₈H₁₁₁N₁₂O₁₆: 1471.8241. found 1471.8190.

(3) Synthesis of Bdev-2 Synthesis of Compound 27

The compound 24 (204 mg, 0.0724 mmol) was dissolved in CH₂Cl₂ (1448 μL),and Et₃N (20.4 μL, 0.145 mmol, 2.0 equiv) and Ac₂O (13.7 μL, 0.145 mmol,2.0 equiv) were added and the mixture was stirred at room temperaturefor 50 minutes. The same post treatment as in synthesis of the compound4 was conducted, to obtain a compound 27 (200 mg, 96.3%).

Synthesis of Bdev-2

To the compound 27 (200 mg, 0.0697 mmol) was added a solution (6970 μL)of TFA/TIS/H₂O=95:5:5 and the mixture was stirred at room temperaturefor 3 hours. The same post treatment as in synthesis of Bdev-7 wasconducted, to obtain Bdev-2 (54.4 mg, 59.8%). HRMS m/z [M+H]⁺: calcd forC₆₆H₈₇N₁₂O₁₆: 1303.6363. found 1303.5524.

(4) Synthesis of Bdev-10

To Bdev-2 (13.1 mg, 0.0101 mmol) and SUNBRIGHT® ME-020AS (manufacturedby NOF Corporation) (40.4 mg, 0.0202 mmol, 2.0 equiv) in DMF (404 μL)was added Et₃N (2.84 μL, 0.0202 mmol, 2.0 equiv), and the mixture wasstirred at room temperature for 22 hours and 40 minutes. Purification byHPLC was performed, to obtain Bdev-10 (16.8 mg, 48.9%).

The structure of the targeted substance was identified based ondetection of divalent ions at 22-amu interval around m/z 1700 by MSmeasurement.

Example 8 Synthesis of Bdev-14

The synthesis route of Bdev-14 is shown schematically in FIG. 3.

Bdev-14 was synthesized as described below.

Synthesis of Compound 88

A mixture of the compound 25 (209 mg, 0.0739 mmol), MeO-PEG-CO₂H[manufactured by Iris Biotech, MW 2,000 Da] (299 mg, 0.149 mmol, 2.0equiv), HOAt (58.5 mg, 0.430 mmol, 5.8 equiv), DIPCI (57.6 μL, 0.370mmol, 5.0 equiv) and DIPEA (64.4 μL, 0.370 mmol, 5.0 equiv) in THF (1478μL) was stirred at room temperature for 3 hours and 50 minutes.MeO-PEG-CO₂H (297 mg, 0.148 mmol, 2.0 equiv), HOAt (60.9 mg, 0.447 mmol,6.0 equiv) and DIPCI (57.6 μL, 0.370 mmol, 5.0 equiv) were added and themixture was stirred at room temperature for 40 minutes. The solvent wasevaporated under reduced pressure, and the residue was subjected tosilica gel column chromatography (CHCl₃/MeOH, 100:0-90:10), to obtain acompound 88 (673 mg) as a crude product.

Synthesis of Bdev-14

To the compound 88 (673 mg) was added a solution (7390 μL) ofTFA/TIS/H₂O=95:5:5 and the mixture was stirred at room temperature for 3hours. The precipitated material was filtrated, and the filtrate wasevaporated under reduced pressure. The residue was purified by GPC, toobtain Bdev-14 (5.6 mg, 2.3%; 2 steps from compound 25). The structureof the targeted substance was identified since a group of peaks at22-amu interval observed ranging from m/z 1400 to 1800 around m/z 1600corresponded to a group of divalent ions derived from the PEG compoundand a group of peaks at 14-amu interval observed ranging from m/z 1000to 1200 around m/z 1100 corresponded to a group of tri-valent ionsderived from the PEG compound, respectively, by MS measurement.

Example 9 Synthesis of Bdev-19

The synthesis route of Bdev-19 is shown schematically in FIG. 4 and FIG.5.

Bdev-19 was synthesized as described below.

Synthesis of Compound 41

A compound 41 was obtained (q.y.) in the same manner as in theabove-described synthesis example of the compound 1, excepting that theamino acid to be condensed was changed to Fmoc-Gln(Trt)-OH.

Synthesis of Compound 42

The compound 41 (13.50 g, 10.0 mmol) was dissolved in THF (180 mL) andDMF (20 mL), and piperidine (2000 μL) and DBU (2000 μL) were added andthe mixture was stirred at room temperature for 5 minutes. Concentratedhydrochloric acid was added until pH of the reaction solution reachedaround 6, and the solvent was evaporated under reduced pressure. To theresidue was added acetonitrile (540 mL) to find deposition of aprecipitated material, which was then filtrated, and suspended andwashed with acetonitrile twice, to obtain a de-Fmoc form.

The de-Fmoc form was dissolved in THF (140 mL) and DMF (60 mL), andFmoc-Ser(tBu)-OH (4.60 g, 12.0 mmol), HOBt.H₂O (1.84 g, 12.0 mmol), HBTU(4.55 g, 12.0 mmol) and DIPEA (8709 μL, 60.0 mmol) were added and themixture was stirred at room temperature for 30 minutes. The solvent wasevaporated under reduced pressure, then, to the residue was addedacetonitrile (700 mL) to find deposition of a precipitated material,which was then filtrated, and suspended and washed with acetonitriletwice, to obtain a compound 42 (14.60 g, 97.8%).

Synthesis of Compound 43

A crude product obtained by treating in the same manner as in theabove-described synthesis example of the compound 42 was purified bynormal phase silica gel column chromatography (toluene/THF=100:0→90:10),to obtain a compound 43 (12.6 g, 72.6%), excepting that the amino acidto be condensed was changed to Fmoc-Trp(Boc)-OH.

Synthesis of Compound 91

The compound 43 (2.60 g, 14.6 mmol) was dissolved in DCM (40 mL) and TFE(4 mL), and TFA (39 μL) was added and the mixture was stirred at roomtemperature for 15 minutes. The precipitated material was filtrated,then, to the filtrate was added water (8 mL) and the mixture wasevaporated under reduced pressure. The generated precipitate wascentrifugally separated (3500 rpm, 6 min), and the precipitated materialwas dissolved in THF and EtOH, then, evaporated under reduced pressure.The residue was suspended and washed with IPE twice, and dried in vacuo,to obtain a compound 91 (98.6%).

Synthesis of Compound 79

A mixture of the compound 29 (1.35 g, 1.15 mmol), the compound 33 (963mg, 1.74 mmol), HATU (656 mg, 1.73 mmol, 1.5 equiv), HOAt (235 mg, 1.73mmol, 1.5 equiv) and DIPEA (1002 μL, 5.75 mmol, 5.0 equiv) in THF (23mL) was stirred at room temperature for 2 hours and 30 minutes. The samepost treatment as in synthesis of the compound 4 was conducted, toobtain a compound 79 (2.05 g).

Synthesis of Compound 80

The compound 79 (2.05 g) was dissolved in THF (23 mL), and piperidine(230 μL, 2.16 mmol, 1.9 equiv) and DBU (230 μL, 1.54 mmol, 1.3 equiv)were added and the mixture was stirred at room temperature for 5minutes. The same post treatment as in synthesis of the compound 2 wasconducted, to obtain a de-Fmoc form (1.68 g, 98.3% from compound 29).

The de-Fmoc form (1.68 g, 1.13 mmol) was dissolved in THF (23 mL), andFmoc-Leu-OH (598 mg, 1.69 mmol, 1.5 equiv), HATU (646 mg, 1.70 mmol, 1.5equiv), HOAt (233 mg, 1.71 mmol, 1.5 equiv) and DIPEA (984 μL, 5.65mmol, 5.0 equiv) were added and the mixture was stirred at roomtemperature for 40 minutes. The same post treatment as in synthesis ofthe compound 4 was conducted, to obtain a compound 80 (1.92 g, 95.6%).

Synthesis of Compound 81

A compound 81 was synthesized (95.5% from compound 80) in the samemanner as in the above-described synthesis example of the compound 80,excepting that the amino acid to be condensed was changed toFmoc-Tyr(tBu)-OH.

Synthesis of Compound 82

A de-Fmoc form was obtained (99.2%) in the same manner as in theabove-described synthesis example of the compound 80, excepting that thestarting material was changed to the compound 81.

The de-Fmoc form (1.75 g, 0.966 mmol) was dissolved in THF (20 mL), andthe compound 91 (1.51 g, 1.45 mmol, 1.5 equiv), HATU (551 mg, 1.45 mmol,1.5 equiv), HOAt (197 mg, 1.45 mmol, 1.5 equiv) and DIPEA (841 μL, 4.83mmol, 5.0 equiv) were added and the mixture was stirred at roomtemperature for 30 minutes. DIPCI (150 μL, 0.963 mmol, 1.0 equiv) andHOAt (197 mg, 1.45 mmol, 1.5 equiv) were added and the mixture wasstirred for 30 minutes. The same post treatment as in synthesis of thecompound 4 was conducted, to obtain a compound 82 (2.60 g, 98.2%).

Synthesis of Compound 83

A de-Fmoc form was obtained (97.8%) in the same manner as in theabove-described synthesis example of the compound 80, excepting that thestarting material was changed to the compound 82.

The de-Fmoc form (2.34 g, 0.909 mmol), the compound 90 (2.17 g, 1.80mmol, 2.0 equiv) and PPh₃ (944 mg, 3.60 mmol, 4.0 equiv) were dissolvedin THF (18 mL), and DEAD (1628 μL, 3.59 mmol, 3.9 equiv) was added andthe mixture was stirred at room temperature for 2 hours. The same posttreatment as in synthesis of the compound 4 was conducted to obtain aresidue, which was then subjected to silica gel column chromatography(toluene/THF, 100:0-85:15), to obtain a compound 83 (1.88 g, 54.9%).

Synthesis of Compound 84

A compound 84 was obtained (78.8%) in the same manner as in theabove-described synthesis example of the compound 15, excepting that thestarting material was changed to the compound 83.

Synthesis of Compound 85

The compound 84 (403 mg, 0.134 mmol) was dissolved in THF (1340 μl), andPhSH (138 μL, 1.34 mmol, 10 equiv) and DBU (200 μL, 1.34 mmol, 10 equiv)were added and the mixture was stirred at room temperature for 50minutes. To the reaction solution was added concentrated hydrochloricacid (112 μl), then, the same post treatment as for synthesis of thecompound 5 was conducted, to obtain a compound 85 (343 mg, 90.3%).

Synthesis of Compound 86

A compound 86 was obtained (144 mg, 82.8%) in the same manner as in theabove-described synthesis example of the compound 22, excepting that thestarting, material was changed to the compound 85.

Synthesis of Bdev-19

Bdev-19 was obtained in the same manner as in the above-describedsynthesis example of Bdev-5, excepting that the starting material waschanged to the compound 86. HRMS m/z [M+H]⁺: calcd for C₆₆H₈₇N₁₂O₁₆:1303.64. found 1303.87.

Example 10 Synthesis of Bdev-20

The synthesis route of Bdev-20 is shown schematically in FIG. 5.

Bdev-20 was synthesized as described below.

Synthesis of Compound 87

A compound 87 (340 mg) was obtained as a crude product in the samemanner as in the above-described synthesis example of the compound 21,excepting that the starting material was changed to the compound 85 (172mg).

Synthesis of Bdev-20

Bdev-20 was obtained (7.8%; 2 steps form compound 85) in the same manneras in the above-described synthesis example of Bdev-3, excepting thatthe starting material was changed to the compound 87. The structure ofthe targeted substance was identified since a group of peaks at 22-amuinterval observed ranging from m/z 1400 to 1800 around m/z 1600corresponded to a group of divalent ions derived from the PEG compoundand a group of peaks at 14-amu interval observed ranging from m/z 1000to 1200 around m/z 1060 corresponded to a group of tri-valent ionsderived from the PEG compound, respectively, by MS measurement.

Example 11 Synthesis of Bdev-21

The synthesis route of Bdev-21 is shown schematically in FIG. 6.

Bdev-21 was synthesized as described below.

Synthesis of Compound 89

The compound 5 (10.9 g, 9.69 mmol) was dissolved in THF (200 mL), andBoc-Tyr(tBu)-OH (4.90 g, 14.5 mmol, 1.5 equiv), HATU (5.53 g, 14.5 mmol,1.5 equiv), HOAt (1.98 g, 14.6 mmol, 1.5 equiv) and DIPEA (8439 μL, 48.5mmol, 5.0 equiv) were added and the mixture was stirred at roomtemperature for 1 hour and 30 minutes. Boc-Tyr(tBu)-OH (1.63 g, 4.83mmol, 0.5 equiv) and HOAt (664 mg, 4.88 mmol, 0.5 equiv) were added andthe mixture was stirred for 1 hour and 10 minutes. The solvent wasevaporated under reduced pressure, and to the residue was added CH₃CN tofind deposition of a precipitated material, which was then filtrated,and suspended and washed with CH₃CN twice to obtain a residue, which wasthen subjected to silica gel column chromatography(n-hexane/EtOAc=100:0-85:15), to obtain a compound 89 (11.0 g, 78.6%).

Synthesis of Compound 90

The compound 89 (11.0 g, 7.62 mmol) was dissolved in THF (70 mL), andTBAF (1.0 M solution in THF, 30.0 mL, 30.0 mmol, 3.9 equiv) was addedand the mixture was stirred at room temperature for 13 hours. To thereaction solution was added CH₂Cl₂ and the mixture was washed with asaturated NH₄Cl aqueous solution, water and saturated saline, and theorganic layer was dried over anhydrous MgSO₄, filtrated, then, thefiltrate was evaporated under reduced pressure. The residue wassubjected to silica gel column chromatography (toluene/THF,100:0-85:15), to obtain a compound 90 (3.89 g, 51.0%).

Synthesis of Compound 92

The compound 90 (259 mg, 0.215 mmol), NsNH₂ (104 mg, 0.514 mmol, 2.4equiv) and PPh₃ (105 mg, 0.400 mmol, 1.9 equiv) were dissolved in THF (2mL), and DEAD (181 μL, 0.399 mmol, 1.9 equiv) was added and the mixturewas stirred at room temperature for 3 hours and 55 minutes. PPh₃ (13.3mg, 0.0507 mmol, 0.24 equiv) and DEAD (22.7 μL, 0.0501 mmol, 0.24 equiv)were added and the mixture was stirred for 45 minutes. The same posttreatment as in synthesis of the compound 4 was conducted, to obtain acompound 92 (293 mg, 98.1%).

Synthesis of Compound 93

The compound 13 (884 mg, 0.369 mmol), the compound 92 (536 mg, 0.385mmol, 1.04 equiv) and PPh₃ (197 mg) were dissolved in THF (7.4 mL), andDEAD (168 μL) was added and the mixture was stirred at room temperaturefor 30 minutes. Further, PPh₃ (97.7 mg, 0.372 mmol, 1.0 equiv) and DEAD(84.0 μL, 0.185 mmol, 0.5 equiv) were added and the mixture was stirredfor 30 minutes. Further, PPh₃ (97.6 mg, 0.372 mmol, 1.0 equiv) and DEAD(84.0 μL, 0.185 mmol, 0.5 equiv) were added and the mixture was stirredfor 1 hour and 50 minutes. Further, PPh₃ (97.9 mg, 0.373 mmol, 1.0equiv) and DEAD (84.0 μL, 0.185 mmol, 0.5 equiv) were added and themixture was stirred for 40 minutes. Further, PPh₃ (98.3 mg, 0.375 mmol,1.0 equiv) and DEAD (84.0 μL, 0.185 mmol, 0.5 equiv) were added and themixture was stirred for 30 minutes. The same post treatment as insynthesis of the compound 4 was conducted to obtain a residue, which wasthen subjected to silica gel column chromatography (CH₂Cl₂/THF,100:0-90:10), to obtain a compound 93 (886 mg, 63.7%).

Synthesis of Compound 94

A compound 94 was obtained (68.0% from compound 93) in the same manneras in the above-described synthesis example of the compound 84,excepting that the starting material was changed to the compound 93.

Synthesis of Compound 95

A compound 95 was obtained (110 mg, 82.6%) in the same manner as in theabove-described synthesis example of the compound 85, excepting that thestarting material was changed to the compound 94.

Synthesis of Compound 96

A compound 96 was obtained (76.4%) in the same manner as in theabove-described synthesis example of the compound 86, excepting that thestarting material was changed to the compound 95.

Synthesis of Bdev-21

Bdev-21 was obtained (27.0%) in the same manner as in theabove-described synthesis example of Bdev-19, excepting that thestarting material was changed to the compound 95. HRMS m/z [M+H]⁺: calcdfor C₆₆H₈₇N₁₂O₁₆: 1303.6363. found 1303.5704.

Example 12 Synthesis of Bdev-25

The synthesis route of Bdev-25 is shown schematically in FIG. 7.

Bdev-25 was synthesized as described below.

Synthesis of Compound 97

A mixture of the compound 29 (2.35 g, 2.02 mmol), Fmoc-Nle(6-OH)—OH (890mg, 2.41 mmol, 1.2 equiv), DMT-MM (860 mg, 3.11 mmol, 1.5 equiv) andDIPEA (697 μL, 4.00 mmol, 2.0 equiv) in THF (40 mL) was stirred at roomtemperature for 40 minutes. The same post treatment as in synthesis ofthe compound 4 was conducted, to obtain a compound 97 (2.97 g; 99.0%).

Synthesis of Compound 98

A compound 98 was obtained (89.6%) in the same manner as in theabove-described synthesis example of the compound 80, excepting that thecompound 97 was used as the starting material and the amino acid to becondensed was changed to Fmoc-Leu-OH.

Synthesis of Compound 99

A compound 99 was obtained (2.65 g, 96.6%) in the same manner as in theabove-described synthesis example of the compound 81, excepting that thecompound 98 was used as the starting material and the amino acid to becondensed was changed to Fmoc-Tyr(tBu)-OH.

Synthesis of Compound 100

A compound 100 was obtained (97.1%) in the same manner as in theabove-described synthesis example of the compound 82, excepting that thecompound 99 was used as the starting material.

Synthesis of Compound 101

The compound 100 (3.60 g, 1.37 mmol) was dissolved in THF (30 mL), andpiperidine (300 μL) and DBU (300 μL) were added and the mixture wasstirred at room temperature for 5 minutes. The same post treatment as insynthesis of the compound 2 was conducted, to obtain a compound 101(3.20 g, 97.8%).

Synthesis of Compound 102

The compound 101 (3.20 g, 1.34 mmol), the compound 40 (4.67 g, 3.36mmol, 2.5 equiv) and PPh₃ (1.41 g, 5.36 mmol, 4.0 equiv) were dissolvedin THF (27 mL), and DEAD (2430 μL, 5.36 mmol, 4.0 equiv) was added andthe mixture was stirred at room temperature for 45 minutes. The samepost treatment as in synthesis of the compound 4 was conducted to obtaina residue, which was then subjected to silica gel column chromatography(toluene/THF, 100:0-80:20), to obtain a compound 102 (2.51 g, 49.7%).

Synthesis of Compound 103

A compound 103 was obtained (93.5%) in the same manner as in theabove-described synthesis example of the compound 14, excepting that thecompound 102 was used as the starting material.

Synthesis of Compound 104

A compound 104 was obtained (93.7%) in the same manner as in theabove-described synthesis example of the compound 15, excepting that thecompound 103 was used as the starting material.

Synthesis of Compound 105

A compound 105 was obtained (92.8%) in the same manner as in theabove-described synthesis example of the compound 16, excepting that thecompound 104 was used as the starting material.

Synthesis of Compound 106

A compound 106 was obtained (86.6%) in the same manner as in theabove-described synthesis example of the compound 22, excepting that thecompound 105 was used as the starting material.

Synthesis of Bdev-25

Bdev-25 was obtained (27.1%) in the same manner as in theabove-described synthesis example of Bdev-5, excepting that the compound106 was used as the starting material. HRMS m/z [M+H]⁺: calcd forC₆₆H₈₇N₁₂O₁₆: 1303.64. found 1303.72.

Example 13 Synthesis of Bdev-27, 28, 29 and 30

The synthesis route of Bdev-27, 28, 29 and 30 is shown schematically inFIG. 8.

Bdev-27, 28, 29 and 30 were synthesized as described below.

Synthesis of Compound 108

The compound 25 (319 mg, 0.113 mmol) was dissolved in CH₂Cl₂ (2 mL), andEt₃N (31.8 μL, 0.226 mmol, 2.0 equiv) and propionic anhydride (29.1 μL,0.226 mmol, 2.0 equiv) were added and the mixture was stirred at roomtemperature for 45 minutes. The same post treatment as in synthesis ofthe compound 4 was conducted, to obtain a compound 108 (301 mg, 92.9%).

Synthesis of Compound 109

A compound 109 was obtained (90.7%) in the same manner as in theabove-described synthesis example of the compound 108, excepting thatn-valeric anhydride was used instead of propionic anhydride.

Synthesis of Compound 110

The compound 25 (315 mg, 0.111 mmol), MeO—[(CH₂)₂O]₃CH₂CH₂CO₂H(manufactured by Chempep: 31.3 mg, 0.132 mmol, 1.2 equiv), HOAt (18.3mg, 0.134 mmol, 1.2 equiv) and HATU (55.2 mg, 0.145 mmol, 1.3 equiv)were dissolved in THF (2220 μL), and DIPEA (96.7 μL, 0.555 mmol, 5.0equiv) was added and the mixture was stirred at room temperature for 2hours and 10 minutes. The same post treatment as in synthesis of thecompound 4 was conducted, to obtain a compound 110 (176 mg, 52.2%).

Synthesis of Compound 111

A compound III was obtained (40.9%) in the same manner as in theabove-described synthesis example of the compound 110, excepting thatMeO—[(CH₂)₂O]₇CH₂CH₂CO₂H was used instead of MeO—[(CH₂)₂O]₃CH₂CH₂CO₂H.

Synthesis of Bdev-27

To the compound 108 (301 mg, 0.105 mmol) were added a solution (10 mL)of TFA/TIS/H₂O=95:5:5 and TIS (1 mL) and the mixture was stirred at roomtemperature for 4 hours and 30 minutes. The same post treatment as insynthesis of Bdev-7 was conducted, to obtain Bdev-27 (28.6 mg, 20.7%).HRMS m/z [M+H]⁺: calcd for C₆₇H₈₉N₁₂O₁₆: 1317.65. found 1317.52.

Synthesis of Bdev-28

Bdev-28 was obtained (33.7%) in the same manner as in theabove-described synthesis example of Bdev-27, excepting that thecompound 109 was used as the starting material. HRMS m/z [M+H]⁺: calcdfor C₆₉H₉₃N₁₂O₁₆: 1345.68. found 1345.58.

Synthesis of Bdev-29

Bdev-29 was obtained (18.1%) in the same manner as in theabove-described synthesis example of Bdev-27, excepting that thecompound 110 was used as the starting material. HRMS m/z [M+H]⁺: calcdfor C₇₄H₁₀₃N₁₂O₂₀: 1479.74. found 1479.57.

Synthesis of Bdev-30

Bdev-30 was obtained (4.9%) in the same manner as in the above-describedsynthesis example of Bdev-27, excepting that the compound III was usedas the starting material. HRMS m/z [M+H]⁺: calcd for C₈₂H₁₁₉N₁₂O₂₄:1655.85. found 1655.67.

Example 14 Synthesis of Bdev-31

The synthesis route of Bdev-31 is shown schematically in FIG. 9.

Bdev-31 was synthesized as described below.

Synthesis of Compound 112

A compound 112 was obtained (q. y.) in the same manner as in theabove-described synthesis example of the compound 1, excepting that theamino acid to be condensed was changed to Fmoc-Asp(OtBu)-OH.

Synthesis of Compound 113

A compound 113 was obtained (q. y.) in the same manner as in theabove-described synthesis example of the compound 42, excepting that thecompound 112 was used as the starting material and the amino acid to becondensed was changed to Fmoc-Gly-OH.

Synthesis of Compound 114

A compound 114 was obtained (15.9 g, 98.1%, 5 steps from Kb-OH) in thesame manner as in the above-described synthesis example of the compound42, excepting that the compound 113 was used as the starting materialand the amino acid to be condensed was changed to Fmoc-Arg(Pbf)-OH.

Synthesis of Compound 115

A compound 115 was obtained (87.0%) in the same manner as in theabove-described synthesis example of the compound 91, excepting that thecompound 114 was used as the starting material.

Synthesis of Compound 116

The compound 32 (2.32 g, 1.60 mmol) was dissolved in THF (32 mL), andpiperidine (238 μL, 2.24 mmol, 1.4 equiv) and DBU (321 μL, 2.15 mmol,1.3 equiv) were added and the mixture was stirred at room temperaturefor 5 minutes. The same post treatment as in synthesis of the compound 2was conducted, to obtain a de-Fmoc form (2.01 g, 99.0%).

The de-Fmoc form (2.01 g, 1.59 mmol) was dissolved in THF (29 mL) andDMF (3.2 mL), and Fmoc-D-Phe-OH (924 mg, 2.38 mmol, 1.5 equiv), DMT-MM(904 mg, 3.27 mmol, 2.1 equiv) and DIPEA (277 μL, 1.59 mmol, 1.0 equiv)were added and the mixture was stirred at room temperature for 30minutes, then, evaporated under reduced pressure. The same posttreatment as in synthesis of the compound 4 was conducted, to obtain acompound 116 (2.50 g, 98.4%).

Synthesis of Compound 117

The compound 116 (2.50 g, 1.56 mmol) was dissolved in THF (31 mL), andpiperidine (232 μL, 2.18 mmol, 1.4 equiv) and DBU (313 μL, 2.09 mmol,1.3 equiv) were added and the mixture was stirred at room temperaturefor 5 minutes. The same post treatment as in synthesis of the compound 2was conducted, to obtain a compound 117 (2.26 g, q. y.).

Synthesis of Compound 118

The compound 117 (2.16 g, 1.53 mmol) was dissolved in THF (28 mL) andDMF (3 mL), and the compound 115 (2.02 g, 2.30 mmol, 1.5 equiv), DMT-MM(918 g, 3.32 mmol, 2.2 equiv) and DIPEA (1334 μL, 7.66 mmol, 5.0 equiv)were added and the mixture was stirred at room temperature for 30minutes, then, evaporated under reduced pressure. The same posttreatment as in synthesis of the compound 4 was conducted, to obtain acompound 118 (3.41 g, 99.7%).

Synthesis of Compound 119

The compound 118 (3.41 g, 1.53 mmol) was dissolved in THF (31 mL), andpiperidine (227 μL, 2.13 mmol, 1.4 equiv) and DBU (305 μL, 2.04 mmol,1.3 equiv) were added and the mixture was stirred at room temperaturefor 5 minutes. The same post treatment as in synthesis of the compound 2was conducted, to obtain a de-Fmoc form (3.25 g, q. y.).

The de-Fmoc form (2.05 g, 1.00 mmol) was dissolved in THF (18 mL) andDMF (2 mL), and Fmoc-Nle (6-OH)—OH (443 mg, 1.20 mmol, 1.2 equiv),DMT-MM (381 mg, 1.20 mmol, 1.2 equiv) and DIPEA (348 μL, 2.00 mmol, 2.0equiv) were added and the mixture was stirred at room temperature for 30minutes, then, evaporated under reduced pressure. The same posttreatment as in synthesis of the compound 4 was conducted, to obtain acompound 119 (2.43 g, q. y.).

Synthesis of Compound 120

The compound 119 (1.18 g, 0.500 mmol) was dissolved in THF (9 mL) andDMF (1 mL), and piperidine (59.0 μL, 0.554 mmol, 1.1 equiv) and DBU(70.0 μL, 0.468 mmol, 0.94 equiv) were added and the mixture was stirredat room temperature for 5 minutes. The same post treatment as insynthesis of the compound 2 was conducted, to obtain a de-Fmoc form(1.05 g, 96.0%).

The de-Fmoc form (435 mg, 0.200 mmol) was dissolved in CH₂Cl₂ (4 mL),and Boc₂O (65.0 mg, 0.298 mmol, 1.5 equiv) and Et₃N (84.0 μL, 0.598mmol, 3.0 equiv) were added and the mixture was stirred at roomtemperature for 1 hour. Boc₂O (22.5 mg, 0.103 mmol, 0.52 equiv) wasadded and disappearance of starting materials was confirmed, then, thiswas evaporated under reduced pressure. The same post treatment as insynthesis of the compound 4 was conducted, to obtain a compound 120 (481mg, q.y. %).

Synthesis of Compound 121

The compound 120 (218 mg, 0.0973 mmol) and PPh₃ (51.1 mg, 0.195 mmol,2.0 equiv) were dissolved in toluene (28.5 mL), and DEAD (88.0 μL, 0.194mmol, 2.0 equiv) was diluted with toluene (20 mL) and dropped over aperiod of 1 hour and the mixture was stirred at room temperature for 30minutes. The same post treatment as in synthesis of the compound 4 wasconducted to obtain a residue, which was then subjected to silica gelcolumn chromatography (CHCl₃/EtOH, 100:0-90:10), to obtain a compound121 (198 mg, 91.6%).

Synthesis of Compound 122

A compound 122 was obtained (q. y.) in the same manner as in theabove-described synthesis example of the compound 16, excepting that thecompound 121 was used as the starting material.

Synthesis of Bdev-31

Bdev-31 was obtained (77.1%) in the same manner as in theabove-described synthesis example of Bdev-7, excepting that the compound122 was used as the starting material. MS m/z [M+H]⁺: calcd forC₃₃H₅₃N₁₀O₉: 733.40. found 733.39.

Example 15 Synthesis of Bdev-32

The synthesis route of Bdev-32 is shown schematically in FIGS. 10 to 16.

Bdev-32 was synthesized as described below.

Synthesis of Compound 123

The compound 2 (2.55 g, 3.00 mmol) was dissolved in THF (54 mL) and DMF(6 mL), and Fmoc-Lys (Boc)-OH (1.69 g, 3.60 mmol, 1.2 equiv), HBTU (1.37g, 3.60 mmol, 1.2 equiv), HOBt.H₂O (482 mg, 3.15 mmol, 1.05 equiv) andDIPEA (1568 μL, 9.00 mmol, 3.0 equiv) were added and the mixture wasstirred at room temperature for 30 minutes, then, evaporated underreduced pressure. The same post treatment as in synthesis of thecompound 4 was conducted on the residue, to obtain a compound 123 (3.80g, q. y.).

Synthesis of Compound 124

A compound 124 was obtained (97.7%) in the same manner as in theabove-described synthesis example of the compound 42, excepting that thecompound 123 was used as the starting material and the amino acid to becondensed was changed to Fmoc-Ser(tBu)-OH.

Synthesis of Compound 125

A compound 125 was obtained in the same manner as in the above-describedsynthesis example of the compound 42, excepting that the compound 124was used as the starting material and the amino acid to be condensed waschanged to Fmoc-Leu-OH.

Synthesis of Compound 126

A compound 126 was obtained (83.7%, 7 steps from compound 2) in the samemanner as in the above-described synthesis example of the compound 42,excepting that the compound 125 was used as the starting material andthe amino acid to be condensed was changed to Boc-Gly-OH.

Synthesis of Compound 127

The compound 126 (3.66 g, 2.51 mmol) was dissolved in CH₂Cl₂ (126 mL),and TFE (13 mL) and TFA (1300 μL) were added and the mixture was stirredat room temperature for 30 minutes. The precipitated material wasfiltrated, to the filtrate was added DIPEA (2940 μL, 16.7 mmol), then,the mixture was evaporated under reduced pressure. To the residue wasadded water to find deposition of a precipitated material, which wasthen filtrated, and suspended and washed with water twice, to obtain acompound 127 (1.53 g, 84.9%).

Synthesis of Compound 128

A compound 128 was obtained (96.5%) in the same manner as in theabove-described synthesis example of the compound 123, excepting thatthe amino acid to be condensed was changed to Fmoc-Phe-OH.

Synthesis of Compound 129

A compound 129 was obtained (99.3%) in the same manner as in theabove-described synthesis example of the compound 42, excepting that thecompound 128 was used as the starting material and the amino acid to becondensed was changed to Fmoc-Nle(6-OH)—OH.

Synthesis of Compound 130

A compound 130 was obtained (84.6%) in the same manner as in theabove-described synthesis example of the compound 2, excepting that thecompound 129 was used as the starting material.

Synthesis of Compound 131

The compound 130 (564 mg, 0.500 mmol) was dissolved in THF (9 mL) andDMF (1 mL), and the compound 127 (430 mg, 0.600 mmol, 1.2 equiv), DMT-MM(166 mg, 0.600 mmol, 1.2 equiv) and DIPEA (261 μL, 1.50 mmol, 3.0 equiv)were added and the mixture was stirred at room temperature for 30minutes, then, evaporated under reduced pressure. The same posttreatment as in synthesis of the compound 4 was conducted, to obtain acompound 131 (817 mg, 91.3%).

Synthesis of Compounds 132 to 137

Fmoc de-protection reaction: A Fmoc-protected peptide (1.0 equiv) wasdissolved in THF (40 mL), and piperidine (1.3 equiv) and DBU (1.2 equiv)were added and the mixture was stirred at room temperature for 5minutes. The same post treatment as in synthesis of the compound 2 wasconducted, to obtain a de-Fmoc form.

Amino acid condensation reaction: The de-Fmoc form (1.0 equiv) wasdissolved in THF (36 mL) and DMF (4 mL), and Fmoc-amino acid (1.2equiv), HBTU (1.2 equiv), HOBt.H₂O (1.2 equiv) and DIPEA (3.6 equiv)were added and the mixture was stirred at room temperature for 30minutes, then, evaporated under reduced pressure. The same posttreatment as for synthesis of the compound 4 was conducted. Amino acidelongation was performed by the method as described above, to obtain acompound 137 (11.4 g, 86.4%, 14 steps from compound 131).

Synthesis of Compound 138

A compound 138 was obtained (98.4%) in the same manner as in theabove-described synthesis example of the compound 127, excepting thatthe compound 137 was used as the starting material.

Synthesis of Compound 139

A compound 139 was obtained (q. y.) in the same manner as in theabove-described synthesis example of the compound 123, excepting thatthe amino acid to be condensed was changed to Fmoc-Ser(tBu)-OH.

Synthesis of Compound 140

A compound 140 was obtained (99.6%, 3 steps from compound 2) in the samemanner as in the above-described synthesis example of the compound 42,excepting that the compound 139 was used as the starting material andthe amino acid to be condensed was changed to Fmoc-Met-OH.

Synthesis of Compound 141

A compound 141 was obtained (99.9%) in the same manner as in theabove-described synthesis example of the compound 42, excepting that thecompound 140 was used as the starting material.

Synthesis of Compound 142

A compound 142 was obtained (88.4%) in the same manner as in theabove-described synthesis example of the compound 91, excepting that thecompound 141 was used as the starting material.

Synthesis of Compound 143

A compound 143 was obtained (q. y.) in the same manner as in theabove-described synthesis example of the compound 123, excepting thatthe amino acid to be condensed was changed to Fmoc-Leu-OH.

Synthesis of Compound 144

A compound 144 was obtained in the same manner as in the above-describedsynthesis example of the compound 2, excepting that the compound 129 wasused as the starting material.

Synthesis of Compound 145

The compound 144 (1.35 g) was dissolved in THF (25 mL) and DMF (3 mL),and the compound 142 (1.27 g, 1.77 mmol), HATU (639 mg, 1.68 mmol), HOAt(200 mg, 1.47 mmol) and DIPEA (732 μL, 4.20 mmol) were added and themixture was stirred at room temperature for 30 minutes. DIPEA (732 μL,4.20 mmol) was added and disappearance of starting materials wasconfirmed, then, this was evaporated under reduced pressure. The samepost treatment as in synthesis of the compound 4 was conducted on theresidue, to obtain a compound 145 (1.98 g, q. y., 2 steps from compound143).

Synthesis of Compound 146

A compound 146 was obtained (820 mg, q. y.) in the same manner as in theabove-described synthesis example of the compound 91, excepting that thecompound 145 was used as the starting material.

Synthesis of Compound 147

The compound 32 (701 mg, 0.484 mmol), the compound 131 (1.73 g, 0.968mmol, 2.0 equiv) and PPh₃ (254 mg, 0.968 mmol, 2.0 equiv) were dissolvedin THF (36 mL), and DEAD (1097 μL, 2.42 mmol, 5.0 equiv) was dissolvedin THF (12 mL) and added over a period of 1 hour and the mixture wasstirred at room temperature for 30 minutes. The same post treatment asin synthesis of the compound 4 was conducted to obtain a residue, whichwas then subjected to silica gel column chromatography (CHCl₃/THF,100:0-75:25 and CHCl₃/THF, 100:0-80:20), to obtain a compound 147 (224mg, 14.4%).

Synthesis of Compound 148

A compound 148 was obtained (57.1%) in the same manner as in theabove-described synthesis example of the compound 2, excepting that thecompound 145 was used as the starting material.

Synthesis of Compound 149

The compound 148 (120 mg, 0.0430 mmol) was dissolved in THF (776 μL) andDMF (86.0 μL), and the compound 146 (65.6 mg, 0.0741 mmol, 1.7 equiv),DMT-MM (17.1 mg, 0.0618 mmol, 1.4 equiv) and DIPEA (15.0 μL, 0.0861mmol, 2.0 equiv) were added and the mixture was stirred at roomtemperature for 45 minutes. The mixture was stirred at 40° C. anddisappearance of starting materials was confirmed, then, this wasevaporated under reduced pressure. The same post treatment as insynthesis of the compound 4 was conducted, to obtain a compound 149 (138mg, 84.2%).

Synthesis of Compound 150

A compound 150 was obtained (96.1%) in the same manner as in theabove-described synthesis example of the compound 2, excepting that thecompound 149 was used as the starting material.

Synthesis of Compound 151

The compound 150 (126 mg, 0.0350 mmol) was dissolved in THF (1.25 mL)and DMF (139 μL), and the compound 138 (75.6 mg, 0.0523 mmol, 1.5equiv), DMT-MM (13.2 g, 0.0477 mmol, 1.4 equiv) and DIPEA (24.2 μL,0.139 mmol, 4.0 equiv) were added and the mixture was stirred at roomtemperature for 35 minutes, then, this was evaporated under reducedpressure. The same post treatment as in synthesis of the compound 4 wasconducted, to obtain a compound 151 (164 mg, 94.3%).

Synthesis of Compound 152

The compound 151 (164 mg, 0.0326 mmol) was dissolved in THF (1.8 mL) andDMF (196 μl), and piperidine (3.90 μl, 0.0366 mmol, 1.1 equiv) and DBU(9.20 μL, 0.0615 mmol, 1.9 equiv) were added and the mixture was stirredat room temperature for 5 minutes. DBU (3.00 μL, 0.0201 mmol) was addedand disappearance of raw materials was confirmed, then, the same posttreatment as in synthesis of the compound 2 was conducted, to obtain ade-Fmoc form (191 mg).

The de-Fmoc form was dissolved in CH₂Cl₂ (3.3 mL), and TEE (326 μL) andTEA (32.6 μL) were added and the mixture was stirred at room temperaturefor 15 minutes. The precipitated material was filtrated, to the filtratewas added DIPEA (76.5 μL, 0.439 mmol), then, this was evaporated underreduced pressure. To the residue was added water to find deposition of aprecipitated material, which was then filtrated, and suspended andwashed with water twice, to obtain a de-Kb form (114 mg).

The de-Kb form was dissolved in THF (5.0 mL) and DMF (560 μL), andDMT-MM (11.1 mg, 0.0401 mmol) and DIPEA (9.80 μL, 0.0563 mmol) wereadded and the mixture was stirred at room temperature for 2 hours, then,this was evaporated under reduced pressure. The same post treatment asin synthesis of the compound 4 was conducted, to obtain a compound 152(110 mg, 83.7%, 3 steps from compound 151).

Synthesis of Compound 153

A compound 153 was obtained (98.2%) in the same manner as in theabove-described synthesis example of the compound 16, excepting that thecompound 152 was used as the starting material.

Synthesis of Bdev-32

To the compound 16 (103 mg, 0.0268 mmol) was added a solution (3 mL) ofTFA/H₂O/PhOH/PhSMe/EDT=82.5/5/5/5/2.5 and the mixture was stirred atroom temperature for 6 hours. The same post treatment as in synthesis ofBdev-7 was conducted, to obtain Bdev-32 (43.0 mg, 76.4%) as a crudeproduct. MS m/z [M+H]⁺: calcd for C₉₉H₁₇₁ N₂₈O₂₈S: 2232.25. found2232.23.

Example 16 Synthesis of Bdev-33

The synthesis route of Bdev-33 is shown schematically in FIGS. 17 to 20.

Bdev-33 was synthesized as described below.

Synthesis of Compound 154

A compound 154 was obtained (q. y.) in the same manner as in theabove-described synthesis example of the compound 1, excepting that theamino acid to be condensed was Fmoc-Leu-OH.

Synthesis of Compound 155

A compound 155 was obtained (99.8%) in the same manner as in theabove-described synthesis example of the compound 42, excepting that thecompound 154 was used as the starting material and the amino acid to becondensed was changed to Fmoc-Met-OH.

Synthesis of Compound 156

The compound 155 (1.67 g, 1.36 mmol) was dissolved in CH₂Cl₂ (136 mL),and TFE (13.6 mL) and TFA (1361 μL) were added and the mixture wasstirred at room temperature for 30 minutes. The precipitated materialwas filtrated, to the filtrate was added DIPEA (3191 μL, 18.3 mmol),then, this was evaporated under reduced pressure. To the residue wasadded water for dilution, 1 N HClaq was added to attain pH4, and CH₂Cl₂was added and extraction thereof was performed. The organic layer waswashed with water three times, washed with saturated NaClaq, dried overMgSO₄, filtrated, and the filtrate was evaporated under reducedpressure. The residue was suspended and washed with n-hexane twice, toobtain a compound 156 (664 mg, q. y.).

Synthesis of Compound 157

A compound 157 was obtained (93.1%) in the same manner as in theabove-described synthesis example of the compound 40, excepting that theamino acid to be condensed was Fmoc-Thr(tBu)-OH.

Synthesis of Compound 158

A compound 158 was obtained (92.2%) in the same manner as in theabove-described synthesis example of the compound 42, excepting that thecompound 157 was used as the starting material and the amino acid to becondensed was changed to Fmoc-Ser(tBu)-OH.

Synthesis of Compound 159

A compound 159 was obtained (94.9%) in the same manner as in theabove-described synthesis example of the compound 42, excepting that thecompound 158 was used as the starting material and the amino acid to becondensed was changed to Fmoc-Leu-OH.

Synthesis of Compound 160

A compound 160 was obtained (97.9%) in the same manner as in theabove-described synthesis example of the compound 42, excepting that thecompound 159 was used as the starting material and the amino acid to becondensed was changed to Fmoc-Asn(Trt)-OH.

Synthesis of Compound 161

A compound 161 was obtained (99.7%) in the same manner as in theabove-described synthesis example of the compound 42, excepting that thecompound 160 was used as the starting material and the amino acid to becondensed was changed to Fmoc-Gly-OH.

Synthesis of Compound 162

A de-Fmoc form was obtained in the same manner as in the above-describedsynthesis example of the compound 42, excepting that the compound 161(2.11 g, 0.997 mmol) was used as the starting material.

The de-Fmoc form was dissolved in THF (18 mL) and DMF (2 mL), andFmoc-Nle(6-OH)—OH (443 mg, 1.20 mmol), DMT-MM (381 mg, 1.38 mmol) andDIPEA (348 μL, 2.00 mmol) were added and the mixture was stirred at roomtemperature for 40 minutes, then, evaporated under reduced pressure. Thesame post treatment as in synthesis of the compound 4 was conducted, toobtain a compound 162 (2.29 g, q. y.).

Synthesis of Compound 163

A de-Fmoc form was obtained (1.07 g) in the same manner as in theabove-described synthesis example of the compound 42, excepting that thecompound 162 (1.13 g, 0500 mmol) was used as the starting material.

The de-Fmoc form was dissolved in CH₂Cl₂ (10 mL), and Boc₂O (218 mg,0.999 mmol) and Et₃N (209 μL, 1.49 mmol) were added and the mixture wasstirred at room temperature for 1 hour. Boc₂O (55.0 mg, 0.252 mmol) wasadded and disappearance of starting materials was confirmed, then, thiswas evaporated under reduced pressure. The same post treatment as insynthesis of the compound 4 was conducted, to obtain a compound 163(1.16 g, q. y.).

Synthesis of Compound 164

A compound 164 was synthesized in the same manner as in theabove-described synthesis example of the compound 121, excepting thatthe compound 163 was used as the starting material. The same posttreatment as in synthesis of the compound 4 was conducted to obtain aresidue, which was then subjected to silica gel column chromatography(CH₂Cl₂/THF, 100:0-65:35 and CH₂Cl₂/EtOH, 92:8), to obtain a compound164 (26.9%).

Synthesis of Compound 165

The compound 164 (210 g, 0.0920 mmol) was dissolved in CH₂Cl₂ (10 mL),and TFE (1 mL) and TFA (92.0 μL) were added and the mixture was stirredat room temperature for 15 minutes. The precipitated material wasfiltrated, to the filtrate was added DIPEA (220 μL, 1.26 mmol), then,this was evaporated under reduced pressure. To the residue was addedwater to find deposition of a precipitated material, which was thenfiltrated, and suspended and washed with water twice, suspended andwashed with IPE three times, to obtain a compound 165 (144 mg, q. y.).

Synthesis of Compound 166

A compound 166 was obtained (q. y.) in the same manner as in theabove-described synthesis example of the compound 1, excepting that theamino acid to be condensed was Fmoc-Lys (Hoc)-OH.

Synthesis of Compounds 167 to 175

Fmoc de-protection reaction: A Fmoc-protected peptide (1.0 equiv) wasdissolved in THF (40 mL), and piperidine (1.3 equiv) and DBU (1.2 equiv)were added and the mixture was stirred at room temperature for 5minutes. The same post treatment as in synthesis of the compound 2 wasconducted, to obtain a de-Fmoc form.

Amino acid condensation reaction: The de-Fmoc form (1.0 equiv) wasdissolved in THF (36 mL) and DMF (4 mL), and Fmoc-amino acid (1.2equiv), HBTU (1.2 equiv), HOBt.H₂O (1.2 equiv) and DIPEA (3.6 equiv)were added and the mixture was stirred at room temperature for 30minutes, then, evaporated under reduced pressure. The same posttreatment as for synthesis of the compound 4 was conducted. Amino acidelongation was performed by the method as described above, to obtain acompound 175 (5.03 g, 38.0%, 17 steps from compound 166).

Synthesis of Compound 176

The compound 175 (959 mg, 0.361 mmol) was dissolved in THF (6.8 mL), andthe compound 156 (247 mg, 0.510 mmol, 1.4 equiv), DMT-MM (138 mg, 0.510mmol, 1.4 equiv) and DIPEA (178 μL, 1.02 mmol, 2.8 equiv) were added andthe mixture was stirred at room temperature for 30 minutes, then,evaporated under reduced pressure. The same post treatment as insynthesis of the compound 4 was conducted, to obtain a compound 176(1.13 g, q. y.).

Synthesis of Compound 177

A compound 177 was obtained (91.2%) in the same manner as in theabove-described synthesis example of the compound 2, excepting that thecompound 176 was used as the starting material.

Synthesis of Compound 178

The compound 177 (243 mg, 0.0830 mmol) was dissolved in THF (1.5 mL) andDMF (170 μL), and the compound 165 (136 mg, 0.0993 mmol, 1.2 equiv),DMT-MM (28.0 mg, 0.101 mmol, 1.2 equiv) and DIPEA (28.9 μL, 0.166 mmol,2.0 equiv) were added and the mixture was stirred at room temperaturefor 35 minutes, then, evaporated under reduced pressure. The same posttreatment as in synthesis of the compound 4 was conducted, to obtain acompound 178 (298.2 mg, 85.2%).

Synthesis of Compound 179

A compound 179 was obtained (253 mg, 91.1%) in the same manner as in theabove-described synthesis example of the compound 16, excepting that thecompound 178 was used as the starting material.

Synthesis of Bdev-33

To the compound 179 (127 mg, 0.0316 mmol) was added a solution (3 mL) ofTFA/H₂O/PhOH/PhSMe/EDT=82.5/5/5/5/2.5 and the mixture was stirred atroom temperature for 3 hours. The same post treatment as in synthesis ofBdev-7 was conducted, to obtain Bdev-33 (55.6 mg, 86.7%). MS m/z [M+H]⁺:calcd for C₈₉H₁₄₂N₂₃O₂₉S: 2029.01. found 2028.99.

As shown by these results, it is possible to introduce a cross-linkageof the present invention in place of the original disulfide bond into acyclic peptide showing pharmacological activity, and as a result, apeptide having a novel cross-linkage structure can be synthesized.

Comparative Example Synthesis of Comparative Compound

As a comparative compound, a W9 peptide (disulfide cross-linkage:compound number: STD) was used. The W9 peptide was prepared bycondensing 2,4-docosyloxy benzyl alcohol with an amino acidsuccessively, to synthesize a linear sequence, then, forming a disulfidebond by oxidation with iodine, and cleaving 2,4-docosyloxy benzylalcohol by trifluoroacetic acid.

Reference Example Synthesis of Reference Compound

As a reference example, a modified body in which the cross-link portionhad been converted into a thioether cross-linkage (compound number:Comp. 1) or an olefin cross-linkage (compound number: Comp. 3) was used.The thioether-cross-linked W9 peptide was synthesized according to asynthesis scheme described in FIG. 21. The olefin-cross-linked W9peptide was synthesized according to a synthesis scheme described inFIG. 22.

Example 17 Resistance to Peptidase

Resistance to a peptidase of a peptide of the present invention wasinvestigated using a carboxy peptidase and chymotrypsin. Resistance to apeptidase was measured as described below.

(Decomposition by Carboxy Peptidase)

A carboxy peptidase purchased from SIGMA was treated with PBS to preparean enzyme solution of 1 U/ml, which was then kept warm at 37° C. in awater bath. Then, a peptide solution having a concentration adjusted to5 mg/ml with DMSO: pure water=1:1 mixed solvent was treated with PBS(−)to obtain a concentration of 1 mg/ml, and the enzyme solution and thepeptide solution were mixed at 4:1 so as to give a peptide finalconcentration of 0.2 mg/ml. Thereafter, these were reacted quickly at37° C. With time, the reaction solution was sampled each in an amount of0.1 ml, and a reaction stopping solution (25% TFA in acetonitrile, 20μL) was added to stop the reaction. Samples were analyzed by HPLC, anddecomposition of a peptide by the peptidase was measured. Sampling wasconducted at 0, 0.5 minutes, 1 minute and 2 minutes, and whendecomposition of 50% or more was not obtained at 2 minutes, sampling wasconducted further at 0.5 hours, 1 hour, 3 hours, additionally, sometimesat 6 hours, and for peptides stable for a long period, sampling wasconducted in a range of 24 hours to 168 hours according to demands. Timeat which half of the peptide added was decomposed was measured. Theresults are shown in Table 3.

(Decomposition by Chymotrypsin)

Chymotrypsin purchased from SIGMA was treated with 0.1M Tris-HCl (pH8.0)to prepare an enzyme solution of 4 U/ml, which was then kept warm at 37°C. in a water bath. Then, a peptide solution treated with DMSO: purewater=1:1 to obtain a concentration of 5 mg/ml was treated with 0.1MTris-HCl (pH8.0) to give a concentration of 1 mg/ml, and the enzymesolution and the peptide solution were mixed at 4:1 so as to give apeptide final concentration of 0.2 mg/ml. Thereafter, these were reactedquickly at 37° C. With time, the reaction solution was sampled each inan amount of 0.1 ml, and a reaction stopping solution (25% TFA inacetonitrile, 20 μL) was added to stop the reaction. Samples wereanalyzed by HPLC, and decomposition of a peptide by chymotrypsin wasmeasured. Sampling was conducted at 0, 0.5 minutes, 1 minute and 2minutes, and when decomposition of 50% or more was not obtained at 2minutes, sampling was conducted further at 0.5 hours, 1 hour, 3 hours,and time at which half of the peptide added was decomposed was measured.The results are shown in Table 3.

TABLE 3 half period by half period by compound structure carboxypeptidase chymotrypsin number pattern Z₁ Z₃ Z₂ (t^(1/2)) (t^(1/2))Bdev2  P-1 H Ac OH 18.70 min  1.40 min Bdev3  P-1 Ac PEG₂₀₀₀ OH  >7days5.19 hr Bdev5  P-1 Ac Ac OH  3.34 min  0.39 min Bdev6  P-1 Ac H OH  2.47min  2.09 min Bdev8  P-1 H H OH  2.79 min  1.26 min Bdev10 P-1 PEG₂₀₀₀Ac OH  >7days 58.28 min Bdev13 P-1 Ac Ac PEG₂₀₀₀  >7days 47.41 minBdev-19 P-2 H Ac OH  2.97 min  0.97 min Bdev-20 P-2 H PEG₂₀₀₀ OH 59.60min  3.6 min Bdev-21 P-3 H Ac OH 14.60 min  0.95 min Bdev-25 P-4 H Ac OH 2.63 min  1.85 min Bdev-27 P-1 H —(C═O)-Ethyl OH 14.99 min  4.37 minBdev-28 P-1 H —(C═O)—n-Butyl OH 21.67 min  2.90 min Bdev-29 P-1 H—(C═O)CH₂CH₂(OCH₂)₃OMe OH 15.67 min  3.73 min Bdev-30 P-1 H—(C═O)CH₂CH₂(OCH₂)₇OMe OH 19.22 min  3.66 min STD SS cross-linkage  0.45min  0.40 min Comp. 1 Thioether cross-linkage  0.72 min  0.45 min Comp.3 Olefin cross-linkage  3.29days  2.07 min

The above-described results teach that a W9 peptide mimic of the presentinvention fabricated according to the cross-linking method of thepresent invention shows improved decomposition resistance to apeptidase, as compared with a W9 peptide containing a disulfidecross-linkage or a thioether cross-linkage.

The above detailed descriptions are provided only for explaining theobject and the subject of the present invention, and dot not limit thescope of the appended claims. Various changes and substitutions for thedescribed embodiments are apparent for those skilled in the art based onteachings described in the present specification, without deviating fromthe scope of the appended claims.

INDUSTRIAL APPLICABILITY

The present invention provides a cross-linked peptide containing a novelnonpeptidic cross-linked structure, and a method for synthesizing thesame. Such a cross-linked peptide is useful since it can manifestvarious improved natures.

The invention claimed is:
 1. A cross-linked peptide represented by thefollowing formula P-1:

wherein, X is any one selected from the group consisting of thefollowing formulae X-1 to X-7:

wherein, n represents an integer of 1 to 12, m represents an integer of1 to 24 and l represents an integer of 1 to 24, Y represents an alkylenechain having 1 to 12 carbon atoms or an alkylene chain having 1 to 66carbon atoms containing at least one —O—, —NH— or —S-bond and optionallysubstituted by a divalent oxygen atom or sulfur atom, Z representshydrogen, an optionally substituted alkyl group having 1 to 30 carbonatoms, an optionally substituted acyl group having 1 to 36 carbon atoms,polyethylene glycol, a tBoc group, a Fmoc group, a Cbz group or a Nosylgroup, [E] represents a hydrogen atom, an optionally substituted acylgroup having 1 to 6 carbon atoms or a peptide having 1 to 20 residuescomposed of amino acids or unnatural amino acids as constituentelements, [F] represents a peptide having 1 to 20 residues composed ofamino acids or unnatural amino acids, [G] represents OH, an amino groupor a peptide having 1 to 20 residues composed of amino acids orunnatural amino acids, the sum of the numbers of amino acids of [E], [F]and [G] is at least 3, and R₁, R₃ and R₄ represent each independently ahydrogen atom or a methyl group, and R₂ represents a hydrogen atom or aside chain of an amino acid or unnatural amino acid.
 2. A cross-linkedpeptide represented by the following formula P-1:

wherein, X represents an alkylene chain having 1 to 12 carbon atoms oran alkylene chain having 1 to 66 carbon atoms containing at least one—O— or —S-bond, Y is any one selected from the group consisting of thefollowing compounds Y1-1 to Y1-2, Y2-1 to Y2-4, Y3-1 to Y3-9, Y4-1 toY4-5, and Y5:

wherein, [H] is represented by any one of the following formulae H1 toH3: or

and [I] is represented by any one of the following formulae 11 to 13:

wherein, [J] is represented by any one of the following formulae J1 toJ2:

and [K] is represented by any one of the following formulae K1 to K3:

and o represents an integer of 1 to 12, p represents an integer of 1 to27, q represents an integer of 1 to 24, r represents an integer of 1 to8, s represents an integer of 1 to 16, t represents an integer of 1 to15, u represents an integer of 1 to 11, and W represents 0 or S, Zrepresents hydrogen, an optionally substituted alkyl group having 1 to30 carbon atoms, an optionally substituted acyl group having 1 to 36carbon atoms, polyethylene glycol, a tBoc group, a Fmoc group, a Cbzgroup or a Nosyl group, [E] represents a hydrogen atom, an optionallysubstituted acyl group having 1 to 6 carbon atoms or a peptide having 1to 20 residues composed of amino acids or unnatural amino acids asconstituent elements, [F] represents a peptide having 1 to 20 residuescomposed of amino acids or unnatural amino acids, [G] represents OH, anamino group or a peptide having 1 to 20 residues composed of amino acidsor unnatural amino acids, the sum of the numbers of amino acids of [E],[F] and [G] is at least 3, and R₁, R₃ and R₄ represent eachindependently a hydrogen atom or a methyl group, and R₂ represents ahydrogen atom or a side chain of an amino acid or unnatural amino acid.3. The cross-linked peptide according to claim 1, wherein n representsan integer of 1 to 7, m represents an integer of 1 to 11, and lrepresents an integer of 1 to 12, and Y is any one selected from thegroup consisting of the following compounds Y1-1 to Y1-2, Y2-1 to Y2-4,Y3-1 to Y3-9, Y4-1 to Y4-5, and Y5:

wherein, [H] is represented by any one of the following formulae H1 toH3: or

[I] is represented by any one of the following formulae 11 to 13:

wherein, [J] is represented by any one of the following formulae J1 toJ2:

and [K] is represented by any one of the following formulae K1 to K3:

and wherein o represents an integer of 1 to 8, p represents an integerof 1 to 11, q represents an integer of 1 to 12, r represents an integerof 1 to 8, s represents an integer of 1 to 16, t represents an integerof 1 to 15, u represents an integer of 1 to 11, and W represents 0 or S.4. A cross-linked peptide represented by the following formula P-1:

wherein Z is an acyl group having 1 to 8 carbon atoms, an unsubstitutedor substituted alkyl group having 1 to 8 carbon atoms, polyethyleneglycol having a molecular weight of 100 to 10000 Da represented by—C(═O)—CH₂CH₂ (OCH₂CH₂)_(n)OCH₂CH₂OCH₃ or any one selected from thegroup consisting of the following formulae Z-1 to Z-5:

wherein, n represents an integer of 1 to 12, q′ represents an integer of1 to 12, v represents 1 or 2, w represents an integer of 1 to 12, R₅ isany one selected from the group consisting of the following formulaeR5-1 to R5-5:

and, R₆ is any one selected from the group consisting of the followingformulae R6-1 to R6-5:

X represents an alkylene chain having 1 to 12 carbon atoms or analkylene chain having 1 to 66 carbon atoms containing at least one —O—or —S-bond, Y represents an alkylene chain having 1 to 12 carbon atomsor an alkylene chain having 1 to 66 carbon atoms containing at least one—O—, —NH— or —S-bond and optionally substituted by a divalent oxygenatom or sulfur atom, [E] represents a hydrogen atom, an optionallysubstituted acyl group having 1 to 6 carbon atoms or a peptide having 1to 20 residues composed of amino acids or unnatural amino acids asconstituent elements, [F] represents a peptide having 1 to 20 residuescomposed of amino acids or unnatural amino acids, [G] represents OH, anamino group or a peptide having 1 to 20 residues composed of amino acidsor unnatural amino acids, the sum of the numbers of amino acids of [E],[F] and [G] is at least 3, and R₁, R₃ and R₄ represent eachindependently a hydrogen atom or a methyl group, and R₂ represents ahydrogen atom or a side chain of an amino acid or unnatural amino acid.5. A cross-linked peptide represented by the following formula P-1:

wherein each of [E] and [G] represents a peptide having at least oneamino acid or unnatural amino acid and [F] represents a peptide havingat least two amino acids or unnatural amino acids, X represents analkylene chain having 1 to 12 carbon atoms or an alkylene chain having 1to 66 carbon atoms containing at least one —O— or —S-bond, Y representsan alkylene chain having 1 to 12 carbon atoms or an alkylene chainhaving 1 to 66 carbon atoms containing at least one —O—, —NH— or —S-bondand optionally substituted by a divalent oxygen atom or sulfur atom, Zrepresents hydrogen, an optionally substituted alkyl group having 1 to30 carbon atoms, an optionally substituted acyl group having 1 to 36carbon atoms, polyethylene glycol, a tBoc group, a Fmoc group, a Cbzgroup or a Nosyl group, and R₁, R₃ and R₄ represent each independently ahydrogen atom or a methyl group, and R₂ represents a hydrogen atom or aside chain of an amino acid or unnatural amino acid.
 6. A cross-linkedpeptide represented by the following formula P-2:

wherein X is any one selected from the group consisting of the followingformulae X-1 to X-7:

wherein, n represents an integer of 1 to 12, m represents an integer of1 to 24, and l represents an integer of 1 to 24, Y represents analkylene chain having 1 to 12 carbon atoms or an alkylene chain having 1to 66 carbon atoms containing at least one —O—, —NH— or —S-bond andoptionally substituted by a divalent oxygen atom or sulfur atom, Zrepresents hydrogen, an optionally substituted alkyl group having 1 to30 carbon atoms, an optionally substituted acyl group having 1 to 36carbon atoms, polyethylene glycol, a tBoc group a Fmoc group, a Cbzgroup or a Nosyl group, [E] represents a hydrogen atom, an optionallysubstituted acyl group having 1 to 6 carbon atoms or a peptide having 1to 20 residues composed of amino acids or unnatural amino acids asconstituent elements, [F] represents a peptide having 1 to 20 residuescomposed of amino acids or unnatural amino acids, [G] represents OH, anamino group or a peptide having 1 to 20 residues composed of amino acidsor unnatural amino acids, the sum of the numbers of amino acids of [E],[F] and [G] is at least 3, and R₁, R₃ and R₄ represent eachindependently a hydrogen atom or a methyl group, and R₂ represents ahydrogen atom or a side chain of an amino acid or unnatural amino acid.7. A cross-linked peptide represented by the following formula P-2:

wherein X represents an alkylene chain having 1 to 12 carbon atoms or analkylene chain having 1 to 66 carbon atoms containing at least one —O—or —S-bond, Y is any one selected from the group consisting of thefollowing compounds Y1-1 to Y1-2, Y2-1 to Y2-4, Y3-1 to Y3-9, Y4-1 toY4-5, and Y5:

wherein, [H] is represented by any one of the following formulae H1 toH3: or

and [I] is represented by any one of the following formulae 11 to 13:

wherein, [J] is represented by any one of the following formulae J1 toJ2:

and [K] is represented by any one of the following formulae K1 to K3:

and o represents an integer of 1 to 12, p represents an integer of 1 to27, q represents an integer of 1 to 24, r represents an integer of 1 to8, s represents an integer of 1 to 16, t represents an integer of 1 to15, u represents an integer of 1 to 11, and W represents 0 or S, Zrepresents hydrogen, an optionally substituted alkyl group having 1 to30 carbon atoms, an optionally substituted acyl group having 1 to 36carbon atoms, polyethylene glycol, a tBoc group, a Fmoc group, a Cbzgroup or a Nosyl group, [E] represents a hydrogen atom, an optionallysubstituted acyl group having 1 to 6 carbon atoms or a peptide having 1to 20 residues composed of amino acids or unnatural amino acids asconstituent elements, [F] represents a peptide having 1 to 20 residuescomposed of amino acids or unnatural amino acids, [G] represents OH, anamino group or a peptide having 1 to 20 residues composed of amino acidsor unnatural amino acids, the sum of the numbers of amino acids of [E],[F] and [G] is at least 3, and R₁, R₃ and R₄ represent eachindependently a hydrogen atom or a methyl group, and R₂ represents ahydrogen atom or a side chain of an amino acid or unnatural amino acid.8. The cross-linked peptide according to claim 6, wherein n representsan integer of 1 to 7, m represents an integer of 1 to 11, l representsan integer of 1 to 12, and Y is any one selected from the groupconsisting of the following compounds Y1-1 to Y1-2, Y2-1 to Y2-4, Y3-1to Y3-9, Y4-1 to Y4-5, and Y5:

wherein, [H] is represented by any one of the following formulae H1 toH3: or

and [I] is represented by any one of the following formulae 11 to 13:

wherein, [J] is represented by any one of the following formulae J1 toJ2:

and [K] is represented by any one of the following formulae K1 to K3:

and wherein o represents an integer of 1 to 8, p represents an integerof 1 to 11, q represents an integer of 1 to 12, r represents an integerof 1 to 8, s represents an integer of 1 to 16, t represents an integerof 1 to 15, u represents an integer of 1 to 11, and W represents 0 or S.9. A cross-linked peptide represented by the following formula P-2:

wherein Z is an acyl group having 1 to 8 carbon atoms, an unsubstitutedor substituted alkyl group having 1 to 8 carbon atoms, polyethyleneglycol having a molecular weight of 100 to 10000 Da represented by—C(═O)—CH₂CH₂(OCH₂CH₂)_(n) OCH₂CH₂OCH₃ or any one selected from thegroup consisting of the following formulae Z-1 to Z-5:

wherein n represents an integer of 1 to 12, q′ represents an integer of1 to 12, v represents 1 or 2, w represents an integer of 1 to 12, R₅ isany one selected from the group consisting of the following formulaeR5-1 to R5-5:

and R₆ is any one selected from the group consisting of the followingformulae R6-1 to R6-5:

X represents an alkylene chain having 1 to 12 carbon atoms or analkylene chain having 1 to 66 carbon atoms containing at least one —O—or —S-bond, Y represents an alkylene chain having 1 to 12 carbon atomsor an alkylene chain having 1 to 66 carbon atoms containing at least one—O—, —NH— or —S-bond and optionally substituted by a divalent oxygenatom or sulfur atom, [E] represents a hydrogen atom, an optionallysubstituted acyl group having 1 to 6 carbon atoms or a peptide having 1to 20 residues composed of amino acids or unnatural amino acids asconstituent elements, [F] represents a peptide having 1 to 20 residuescomposed of amino acids or unnatural amino acids, [G] represents OH, anamino group or a peptide having 1 to 20 residues composed of amino acidsor unnatural amino acids, the sum of the numbers of amino acids of [E],[F] and [G] is at least 3, and R₁, R₃ and R₄ represent eachindependently a hydrogen atom or a methyl group, and R₂ represents ahydrogen atom or a side chain of an amino acid or unnatural amino acid.10. A cross-linked peptide represented by the following formula P-2:

wherein each of [E] and [G] represent a peptide having at least oneamino acid or unnatural amino acid and [F] represents a peptide havingat least two amino acids or unnatural amino acids, X represents analkylene chain having 1 to 12 carbon atoms or an alkylene chain having 1to 66 carbon atoms containing at least one —O— or —S-bond, Y representsan alkylene chain having 1 to 12 carbon atoms or an alkylene chainhaving 1 to 66 carbon atoms containing at least one —O—, —NH— or —S-bondand optionally substituted by a divalent oxygen atom or sulfur atom, Zrepresents hydrogen, an optionally substituted alkyl group having 1 to30 carbon atoms, an optionally substituted acyl group having 1 to 36carbon atoms, polyethylene glycol, a tBoc group, a Fmoc group, a Cbzgroup or a Nosyl group, R₁, R₃ and R₄ represent each independently ahydrogen atom or a methyl group, and R₂ represents a hydrogen atom or aside chain of an amino acid or unnatural amino acid.
 11. A method ofsynthesizing a cross-linked peptide represented by the followingformula:

wherein, X and Y represent each independently an alkylene chain having 1to 12 carbon atoms or an alkylene chain having 1 to 66 carbon atomscontaining at least one —O—, —NH— or —S— bond and optionally substitutedby a divalent oxygen atom or sulfur atom, Z represents hydrogen, anoptionally substituted alkyl group having 1 to 30 carbon atoms, anoptionally substituted acyl group having 1 to 36 carbon atoms,polyethylene glycol, a tBoc group, a Fmoc group, a Cbz group or a Nosylgroup, [E] represents a hydrogen atom, an acetyl group or a peptidehaving 1 to 20 residues composed of amino acids or unnatural aminoacids, [G] represents OH, an amino group or a peptide having 1 to 20residues composed of amino acids or unnatural amino acids, [F]represents a peptide having 1 to 20 residues composed of amino acids orunnatural amino acids, the sum of the numbers of amino acids of [E], [F]and [G] is at least 3, R₁, R₃ and R₄ represent each independently ahydrogen atom or a methyl group, and R₂ represents a hydrogen atom or aside chain of an amino acid or unnatural amino acid, comprising thefollowing steps: (a) a step of preparing a first component, comprisingthe following steps, (a-1) a step of reacting the N-terminus side of thepeptide or amino acid synthesized or the carboxyl protective group withan amino acid derivative containing in the side chain a linker formingpart of a cross-linkage of the cross-linked peptide, to synthesize thepeptide having the linker in the side chain or the amino acid derivativehaving the carboxyl group protected, (a-2) when the linker end is notreactive in the following step c of the cross-linkage forming reaction,a step of converting the linker end into a reactive functional group,(b) a step of preparing a second component, comprising the followingsteps, (b-1) a step of condensing a carboxyl protective group with aminoacids or a peptide constituting a partial peptide sequence of thecross-linked peptide, (b-2) a step of reacting the N-terminus of thepeptide or the amino acid derivative synthesized with a compoundcontaining a linker forming part of a cross-linkage of the cross-linkedpeptide, to synthesize a peptide or an amino acid derivative having asecondary amine at the N-terminus containing the linker, (b-3) when thelinker end is not reactive in the following step c of the cross-linkageforming reaction, a step of converting the linker end into a reactivefunctional group, (c) a step of linking the first component and thesecond component by the Mitsunobu reaction, a reductive aminationreaction or the Aza-Wittig reaction and the subsequent reductionreaction, to prepare an intermediate having a structure in which the twocomponents are linked via a secondary amine or a tertiary amine, and (d)a step of condensing the peptide N- or C-terminus of one component withthe peptide C- or N-terminus of another component.
 12. A method ofsynthesizing the cross-linked peptide according to claim 11, wherein thestep (c) is carried out under a condition in which at least one of thefirst component and the second component is linked to the carboxylprotective group of a peptide support, wherein the peptide support is analkoxy substituted benzyl selected from the group consisting of a2,4-substituted benzyl alcohol, a 3,5-substituted benzyl alcohol, a3,4,5-substituted benzyl alcohol and a 2,4,5-substituted benzyl alcohol.13. The method of synthesizing the cross-linked peptide according toclaim 12, wherein the number of carbon atoms of the alkoxy substituentof the 2,4-substituted benzyl alcohol is 1 to 60.